T cell receptors and immune therapy using the same

ABSTRACT

The present invention pertains to antigen recognizing constructs against tumor associated antigens (MAGEA1). The invention in particular provides novel T cell receptor (TCR) based molecules which are selective and specific for the tumor expressed antigen of the invention. The TCR of the invention, and TAA binding fragments derived therefrom, are of use for the diagnosis, treatment and prevention of TAA expressing cancerous diseases. Further provided are nucleic acids encoding the antigen recognizing constructs of the invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen recognizing constructs and pharmaceutical compositions comprising the compounds of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/431,588, filed 8 Dec. 2016, and German Application No.102016123847.3, filed 8 Dec. 2016, the content of each of theseapplications is herein incorporated by reference in their entirety.

This application also is related to PCT/EP2017/081800 filed 7 Dec. 2017,the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-081001_ST25.txt” createdon 6 Dec. 2017, and 128,613 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

BACKGROUND Field

The present invention pertains to antigen recognizing constructs againstthe tumor associated antigen (TAA) derived peptide MAGEA1-003. Theinvention in particular provides novel T cell receptor (TCR) basedmolecules which are selective and specific for the TAA of the invention.The TCR of the invention, and TAA binding fragments derived therefrom,are of use for the diagnosis, treatment and prevention of TAA expressingcancerous diseases. Further provided are nucleic acids encoding theantigen recognizing constructs of the invention, vectors comprisingthese nucleic acids, recombinant cells expressing the antigenrecognizing constructs and pharmaceutical compositions comprising thecompounds of the invention.

Description of Related Art

The melanoma antigen genes (MAGE-A) were found to be expressed in avariety of tumors of different histological origin. Proteins encoded bythe MAGE genes are tumor rejection anti-gens, which can induce specificcytotoxic T-lymphocytes (CTL) having the ability to recognize and killcancerous cells. MAGE genes and proteins are thus a preferential targetfor the development of novel drugs to fight cancer by immunotherapy.MAGE-A proteins constitute a sub-family of Cancer-Testis Antigens whichare expressed mainly, but not exclusively, in the germ line. They arehowever also expressed in various human cancers where they areassociated with, and may drive, malignancy. This specific expression ofMAGE antigens in tumors and not the normal surrounding healthy tissuemakes this family of antigens very interesting for targeted adoptive Tcell transfer. However, to date no satisfactory immune therapy is knowndue to the lack of specific and highly avid antibodies or T cellreceptors targeting the MAGE antigen.

T-cell based immunotherapy targets represent peptide epitopes derivedfrom tumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). These tumorassociated antigens (TAAs) can be peptides derived from all proteinclasses, such as enzymes, receptors, transcription factors, etc. whichare expressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

Specific elements of the cellular immune response are capable ofselectively recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defective ribosomal products (DRiPs) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

There are two classes of MHC-molecules, MHC class I and MHC class II.Complexes of peptide and MHC class I are recognized by CD8-positiveT-cells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T-cells bearing the appropriate TCR. Since bothtypes of response, CD8 and CD4 dependent, contribute jointly andsynergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens and corresponding T cellreceptors is important in the development of cancer immunotherapies suchas vaccines and cell therapies.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T-cells bearingspecific T-cell receptors (TCR). There-fore, TAAs are a starting pointfor the development of a T-cell based therapy including but not limitedto tumor vaccines and cell therapies.

Approximately 90 percent of peripheral blood T cells express a TCRconsisting of an a polypeptide and a β polypeptide. Beside αβ T cells, asmall percentage of T cells (about 5% of total T cells) have been shownto express a TCR consisting of a γ polypeptide and a δ polypeptide. γδ Tcells are found at their highest abundance in the gut mucosa, within apopulation of lymphocytes known as intraepithelial lymphocytes (IELs).The antigenic molecules that activate γδ T cells are still widelyunknown. However, γδ T cells are not MHC restricted and seem to be ableto recognize whole proteins rather than requiring peptides to bepresented by MHC molecules on antigen presenting cells, although somerecognize MHC class IB molecules. Human Vγ9/Vδ2 T cells, whichconstitute the major γδ T cell population in peripheral blood, areunique in that they specifically and rapidly respond to a smallnon-peptidic microbial metabolite, HMB-PP, an isopentenyl pyrophosphateprecursor.

The chains of the T cell antigen receptor of a T cell clone are eachcomposed of a unique combination of domains designated variable (V),[diversity (D),] joining (J), and constant (C). In each T cell clone,the combination of V, D and J domains of both the alpha and the betachains or of both the delta and gamma chains participates in antigenrecognition in a manner which is uniquely characteristic of that T cellclone and defines a unique binding site, also known as the idiotype ofthe T cell clone. In contrast, the C domain does not participate inantigen binding.

A TCR is a heterodimeric cell surface protein of the immunoglobulinsuper-family, which is associated with invariant proteins of the CD3complex involved in mediating signal transduction. TCRs exist in αβ andγδ forms, which are structurally similar but have quite distinctanatomical locations and probably functions. The extracellular portionof native heterodimeric αβTCR and γδTCR each contain two polypeptides,each of which has a membrane-proximal constant domain, and amembrane-distal variable domain. Each of the constant and variabledomains includes an intrα-chain disulfide bond. The variable domainscontain the highly polymorphic loops analogous to the complementaritydetermining regions (CDRs) of antibodies. The use of TCR gene therapyovercomes a number of current hurdles. It allows equipping patients' ownT cells with desired specificities and generation of sufficient numbersof T cells in a short period of time, avoiding their exhaustion. The TCRwill be transduced into central memory T cells or T cells with stem cellcharacteristics, which may ensure better persistence and function upontransfer. TCR-engineered T cells will be infused into cancer patientsrendered lymphopenic by chemotherapy or irradiation, allowing efficientengraftment but inhibiting immune suppression.

SUMMARY

While advances have been made in the development of molecular-targetingdrugs for cancer therapy, there remains a need in the art to develop newanti-cancer agents that specifically target molecules highly specific tocancer cells. The present description addresses that need by providingnovel MAGEA1 TCRs, respective recombinant TCR constructs, nucleic acids,vectors and host cells that specifically bind TAA epitope(s) asdisclosed; and methods of using such molecules in the treatment ofcancer. The term TAA in context of the invention relates in particularto the following preferred proteins, namely MAGEA1 proteins, fragmentsthereof, in particular antigenic peptides presented by HLA, andpreferably associated with a proliferative disorder. The preferredantigenic peptide of the invention is the peptide MAGEA1-003, having theamino acid sequence set forth in any of the SEQ ID NO: 133 to 142 and154 to 162 in table 2 below. A TAA peptide preferably is a peptide asset forth in any of SEQ ID NO: 133 to 142 and 154 to 162.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1-44 depict embodiments of the disclosure as described herein.

FIG. 1: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P1A9 (SEQ ID NO:1-12) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 alanine-substitution variants at positions 1-9 of SEQID NO:1 (SEQ ID NO:134-142) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 2: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P2A6 (SEQ ID NO:13-24) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 alanine-substitution variants at positions 1-9 of SEQID NO:1 (SEQ ID NO:134-142) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 3: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P3H1 (SEQ ID NO:25-36) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 alanine-substitution variants at positions 1-9 of SEQID NO:1 (SEQ ID NO:134-142) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 4: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R35P3A4 (SEQ ID NO:37-48) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 alanine-substitution variants at positions 1-9 of SEQID NO:1 (SEQ ID NO:134-142) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 5: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R37P1C9 (SEQ ID NO:49-60) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 alanine-substitution variants at positions 1-9 of SEQID NO:1 (SEQ ID NO:134-142) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 6: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R37P1H1 (SEQ ID NO:61-72) after co-incubation withT2 target cells loaded with

MAGEA1-003 peptide (SEQ ID NO:133) or various MAGEA1-003alanine-substitution variants at positions 1-9 of SEQ ID NO:1 (SEQ IDNO:134-142) or control peptide NYESO1-001 (SEQ ID NO:153). IFNγ releasedata were obtained with CD8+ T-cells derived from two different donors.RNA electroporated CD8+ T-cells alone or in co-incubation with unloadedtarget cells served as controls. Donor 1 (TCRA-0004) is shown on theleft Y-axis, donor 2 (TCRA-0005) on the right Y-axis, respectively.

FIG. 7: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R42P3A9 (SEQ ID NO:73-84) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 alanine-substitution variants at positions 1-9 of SEQID NO:1 (SEQ ID NO:134-142) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 8: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R43P3F2 (SEQ ID NO:85-96) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 alanine-substitution variants at positions 1-9 of SEQID NO:1 (SEQ ID NO:134-142) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 9: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R43P3G5 (SEQ ID NO:97-108) after co-incubationwith T2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 alanine-substitution variants at positions 1-9 of SEQID NO:1 (SEQ ID NO:134-142) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 10: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R59P2E7 (SEQ ID NO:109-120) after co-incubationwith T2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 alanine-substitution variants at positions 1-9 of SEQID NO:1 (SEQ ID NO:134-142) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 11: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P1A9 (SEQ ID NO:1-12) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orhomologous but unrelated peptide TMEM175-001 (SEQ ID NO:143), EPAS-001(SEQ ID NO:144), PTRF-001 (SEQ ID NO:145), GALNTL4-001 (SEQ ID NO:146),PSME2-001 (SEQ ID NO:147), KIAA103-002 (SEQ ID NO:148), NSD1-001 (SEQ IDNO:149), TBC1D9-002 (SEQ ID NO:150), TMTC4-001 (SEQ ID NO:151) orRASAL2-002 (SEQ ID NO:152) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 12: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P2A6 (SEQ ID NO:13-24) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orhomologous but unrelated peptide TMEM175-001 (SEQ ID NO:143), EPAS-001(SEQ ID NO:144), PTRF-001 (SEQ ID NO:145), GALNTL4-001 (SEQ ID NO:146),PSME2-001 (SEQ ID NO:147), KIAA103-002 (SEQ ID NO:148), NSD1-001 (SEQ IDNO:149), TBC1D9-002 (SEQ ID NO:150), TMTC4-001 (SEQ ID NO:151) orRASAL2-002 (SEQ ID NO:152) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 13: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P3H1 (SEQ ID NO:25-36) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orhomologous but unrelated peptide TMEM175-001 (SEQ ID NO:143), EPAS-001(SEQ ID NO:144), PTRF-001 (SEQ ID NO:145), GALNTL4-001 (SEQ ID NO:146),PSME2-001 (SEQ ID NO:147), KIAA103-002 (SEQ ID NO:148), NSD1-001 (SEQ IDNO:149), TBC1D9-002 (SEQ ID NO:150), TMTC4-001 (SEQ ID NO:151) orRASAL2-002 (SEQ ID NO:152) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 14: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R35P3A4 (SEQ ID NO:37-48) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orhomologous but unrelated peptide TMEM175-001 (SEQ ID NO:143), EPAS-001(SEQ ID NO:144), PTRF-001 (SEQ ID NO:145), GALNTL4-001 (SEQ ID NO:146),PSME2-001 (SEQ ID NO:147), KIAA103-002 (SEQ ID NO:148), NSD1-001 (SEQ IDNO:149), TBC1D9-002 (SEQ ID NO:150), TMTC4-001 (SEQ ID NO:151) orRASAL2-002 (SEQ ID NO:152) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 15: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R37P1C9 (SEQ ID NO:49-60) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orhomologous but unrelated peptide TMEM175-001 (SEQ ID NO:143), EPAS-001(SEQ ID NO:144), PTRF-001 (SEQ ID NO:145), GALNTL4-001 (SEQ ID NO:146),PSME2-001 (SEQ ID NO:147), KIAA103-002 (SEQ ID NO:148), NSD1-001 (SEQ IDNO:149), TBC1D9-002 (SEQ ID NO:150), TMTC4-001 (SEQ ID NO:151) orRASAL2-002 (SEQ ID NO:152) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 16: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R37P1H1 (SEQ ID NO:61-72) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orhomologous but unrelated peptide TMEM175-001 (SEQ ID NO:143), EPAS-001(SEQ ID NO:144), PTRF-001 (SEQ ID NO:145), GALNTL4-001 (SEQ ID NO:146),PSME2-001 (SEQ ID NO:147), KIAA103-002 (SEQ ID NO:148), NSD1-001 (SEQ IDNO:149), TBC1D9-002 (SEQ ID NO:150), TMTC4-001 (SEQ ID NO:151) orRASAL2-002 (SEQ ID NO:152) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 17: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R42P3A9 (SEQ ID NO:73-84) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orhomologous but unrelated peptide TMEM175-001 (SEQ ID NO:143), EPAS-001(SEQ ID NO:144), PTRF-001 (SEQ ID NO:145), GALNTL4-001 (SEQ ID NO:146),PSME2-001 (SEQ ID NO:147), KIAA103-002 (SEQ ID NO:148), NSD1-001 (SEQ IDNO:149), TBC1D9-002 (SEQ ID NO:150), TMTC4-001 (SEQ ID NO:151) orRASAL2-002 (SEQ ID NO:152) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 18: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R43P3F2 (SEQ ID NO:85-96) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orhomologous but unrelated peptide TMEM175-001 (SEQ ID NO:143), EPAS-001(SEQ ID NO:144), PTRF-001 (SEQ ID NO:145), GALNTL4-001 (SEQ ID NO:146),PSME2-001 (SEQ ID NO:147), KIAA103-002 (SEQ ID NO:148), NSD1-001 (SEQ IDNO:149), TBC1D9-002 (SEQ ID NO:150), TMTC4-001 (SEQ ID NO:151) orRASAL2-002 (SEQ ID NO:152) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 19: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R43P3G5 (SEQ ID NO:97-108) after co-incubationwith T2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orhomologous but unrelated peptide TMEM175-001 (SEQ ID NO:143), EPAS-001(SEQ ID NO:144), PTRF-001 (SEQ ID NO:145), GALNTL4-001 (SEQ ID NO:146),PSME2-001 (SEQ ID NO:147), KIAA103-002 (SEQ ID NO:148), NSD1-001 (SEQ IDNO:149), TBC1D9-002 (SEQ ID NO:150), TMTC4-001 (SEQ ID NO:151) orRASAL2-002 (SEQ ID NO:152) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 20: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R59P2E7 (SEQ ID NO:109-120) after co-incubationwith T2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orhomologous but unrelated peptide TMEM175-001 (SEQ ID NO:143), EPAS-001(SEQ ID NO:144), PTRF-001 (SEQ ID NO:145), GALNTL4-001 (SEQ ID NO:146),PSME2-001 (SEQ ID NO:147), KIAA103-002 (SEQ ID NO:148), NSD1-001 (SEQ IDNO:149), TBC1D9-002 (SEQ ID NO:150), TMTC4-001 (SEQ ID NO:151) orRASAL2-002 (SEQ ID NO:152) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0004) is shown on the left Y-axis, donor 2 (TCRA-0005) on theright Y-axis, respectively.

FIG. 21: HLA-A*02/MAGEA1-003 tetramer or HLA-A*02/NYESO1-001 tetramerstaining, respectively, of CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P2A6 and R26P3H1, respectively. CD8+ T-cellselectroporated with RNA of 1G4 TCR that specifically binds toHLA-A*02/NYESO1-001 complex and mock electroporated CD8+ T-cells servedas controls.

FIG. 22: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P1A9 (SEQ ID NO:1-12) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 threonine-substitution variants at positions 1-9 ofSEQ ID NO:1 (SEQ ID NO:154-162) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0006) is shown on the left Y-axis, donor 2 (TCRA-0007) on theright Y-axis respectively.

FIG. 23: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P2A6 (SEQ ID NO:13-24) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 threonine-substitution variants at positions 1-9 ofSEQ ID NO:1 (SEQ ID NO:154-162) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0006) is shown on the left Y-axis, donor 2 (TCRA-0007) on theright Y-axis, respectively.

FIG. 24: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P3H1 (SEQ ID NO:25-36) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 threonine-substitution variants at positions 1-9 ofSEQ ID NO:1 (SEQ ID NO:154-162) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0006) is shown on the left Y-axis, donor 2 (TCRA-0007) on theright Y-axis, respectively.

FIG. 25: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R35P3A4 (SEQ ID NO:37-48) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 threonine-substitution variants at positions 1-9 ofSEQ ID NO:1 (SEQ ID NO:154-162) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0006) is shown on the left Y-axis, donor 2 (TCRA-0007) on theright Y-axis, respectively.

FIG. 26: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R37P1C9 (SEQ ID NO:49-60) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 threonine-substitution variants at positions 1-9 ofSEQ ID NO:1 (SEQ ID NO:154-162) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0006) is shown on the left Y-axis, donor 2 (TCRA-0007) on theright Y-axis, respectively.

FIG. 27: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R37P1H1 (SEQ ID NO:61-72) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 threonine-substitution variants at positions 1-9 ofSEQ ID NO:1 (SEQ ID NO:154-162) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromone donors. RNA electroporated CD8+ T-cells alone or in co-incubationwith unloaded target cells served as controls.

FIG. 28: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R42P3A9 (SEQ ID NO:73-84) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 threonine-substitution variants at positions 1-9 ofSEQ ID NO:1 (SEQ ID NO:154-162) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromone donors. RNA electroporated CD8+ T-cells alone or in co-incubationwith unloaded target cells served as controls.

FIG. 29: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R43P3F2 (SEQ ID NO:85-96) after co-incubation withT2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 threonine-substitution variants at positions 1-9 ofSEQ ID NO:1 (SEQ ID NO:154-162) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0006) is shown on the left Y-axis, donor 2 (TCRA-0007) on theright Y-axis, respectively.

FIG. 30: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R43P3G5 (SEQ ID NO:97-108) after co-incubationwith T2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 threonine-substitution variants at positions 1-9 ofSEQ ID NO:1 (SEQ ID NO:154-162) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0006) is shown on the left Y-axis, donor 2 (TCRA-0007) on theright Y-axis, respectively.

FIG. 31: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R59P2E7 (SEQ ID NO:109-120) after co-incubationwith T2 target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) orvarious MAGEA1-003 threonine-substitution variants at positions 1-9 ofSEQ ID NO:1 (SEQ ID NO:154-162) or control peptide NYESO1-001 (SEQ IDNO:153). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different donors. RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls. Donor 1(TCRA-0006) is shown on the left Y-axis, donor 2 (TCRA-0007) on theright Y-axis, respectively.

FIG. 32: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs R35P3A4 (SEQ ID NO:37-48), R37P1C9 (SEQ IDNO:49-60) and R43P3G5 (SEQ ID NO:97-108) after co-incubation withdifferent tumor cell lines. U266B1 and UACC-257 present the target,KMM-1, NCI-H2023, L-1236, MCF-7 and A-375 do not present the target. T2target cells loaded with MAGEA1-003 or control peptide NYESO1-001 (SEQID NO:153) and RNA electroporated CD8+ T-cells alone served as controls.

FIG. 33: IFNγ release from T-cells after lentiviral transduction withdifferent constructs containing alpha and beta chain of TCRs R35P3A4(SEQ ID NO:37-48; constructs R35D, R35G), R37P1C9 (SEQ ID NO:49-60;constructs R37D, R37G) and R43P3G5 (SEQ ID NO:97-108; constructs R43A,R43H) after co-incubation with different primary cells and iPSC-derivedcell types. U266B1 and UACC-257 and T-cells alone served as controls.

Cell type Abbreviation source Normal Human Dermal Fibroblasts NHDFPrimary cells Human Coronary Artery Smooth HCASMC Primary cells MuscleCells Human Tracheal Smooth Muscle HTSMC Primary cells Cells Human RenalEpithelial Cells HREpC Primary cells Human Cardiomyocytes HCM Primarycells Human Cardiac Microvascular HCMEC Primary cells Endothelial CellsiCell Hepatocytes 2.0 — iPSC-derived cells iCell Astrocytes —iPSC-derived cells iCell Neurons — iPSC-derived cells

FIG. 34: IFNγ release from T-cells after lentiviral transduction withdifferent constructs containing alpha and beta chain of TCR R37P1C9 toassess LV-R37D-mediated recognition of endogenously processed andpresented MAGEA1-003. (A) T cells from three healthy donors,non-transduced (NT) or transduced with LV-R37D were co-cultured with 3different tumor cell lines. IFN-γ production with HLA-A*02+ tumor celllines endogenously expressing MAGEA1 (UACC257 and U266B1) or lackingsurface-presented (KMM1). E:T targets are indicated. (B) IFN-γproduction with serially diluted MAGEA1 peptide pulsed T2 cells.Co-cultures were set up at E:T ratio 4:1 (60,000 T cells; 15,000 T2cells). Mean and standard deviation of IFN-γ released after 20 h fromthree replicates (donors) is shown as duplicate measurements by ELISA.

FIG. 35: Potency assay evaluating cytolytic activity of lentivirallytransduced T cells expressing TCR R37P1C9 (construct R37D) againstMAGEA1+ tumor cells. Cytotoxic response of LV-R37D transduced andnon-transduced (NT) T cells measured against (A) U138MG MAGEA1+ tumorcells (HLA-A*02+), (B) U138MG MAGEA1− (HLA-A*02+), or (C) U2-OSMAGEA1+(HLA-A*02+) tumor cells. The assays were performed at various E:Tratios (A and B) or at 10:1 E:T ratio (C) in a 96 hour fluorescencemicroscopy based cytotoxicity assay. Results are presented as mean±SD of3 replicates. NT—Non-transduced. For graphs A and B, %Killing=(Area-Under-Curve of experiment sample with Tcells/Area-Under-Curve tumor targets alone)*100, where Area-Under-Curveis calculated from the longitudinal growth measurements as shown ingraph C; negative % Killing values are set to 0.

FIG. 36: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P1A9 (Table 1) after co-incubation with T2target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 μM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 37: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R26P2A6 (Table 1) after co-incubation with T2target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 38: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R35P3A4 (Table 1) after co-incubation with T2target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 pM.

FIG. 39: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R37P1C9 (Table 1) after co-incubation with T2target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 40: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R37P1H1 (Table 1) after co-incubation with T2target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 pM.

FIG. 41: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R42P3A9 (Table 1) after co-incubation with T2target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 pM.

FIG. 42: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R43P3F2 (Table 1) after co-incubation with T2target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 43: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R43P3G5 (Table 1) after co-incubation with T2target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

FIG. 44: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R59P2E7 (Table 1) after co-incubation with T2target cells loaded with MAGEA1-003 peptide (SEQ ID NO:133) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Antigen Recognizing Constructs

The object of the invention is solved in a first aspect by an antigenrecognizing construct comprising at least one complementary determiningregion (CDR) 3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or preferably 100% sequence identity to an amino acid sequence selectedfrom SEQ ID NOs. 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81,87, 93, 99, 105, 111 and 117.

In some embodiments the antigen recognizing construct of the inventionspecifically binds to a TAA-peptide-HLA molecule complex, wherein theTAA peptide comprises, or alternatively consists of, a variant of theTAA which is at least 66%, preferably at least 77%, and more preferablyat least 88% homologous (preferably at least 77% or at least 88%identical) to the amino acid sequence of the TAA of the invention,wherein said variant binds to an HLA class I or class II molecule and/orinduces T-cells cross-reacting with said peptide, or a pharmaceuticallyacceptable salt thereof, wherein said peptide is not the underlyingfull-length polypeptide.

As used herein, the terms “identical” or percent “identity”, when usedanywhere herein in the context of two or more nucleic acid orprotein/polypeptide sequences, refer to two or more sequences orsubsequences that are the same or have (or have at least) a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., at, or at least, about 60% identity, preferably at, or at least,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, identity, and morepreferably at, or at least, about 95%, 96%, 97%, 98%, 99%, or higheridentity over a specified region—preferably over their full lengthsequences—, when compared and aligned for maximum correspondence overthe comparison window or designated region) as measured using a sequencecomparison algorithms, or by manual alignment and visual inspection(see, e.g., NCBI web site). In a particular embodiment, for example whencomparing the protein or nucleic acid sequence of an antigen recognizingconstruct of the invention to another protein/gene, the percentageidentity can be determined by the Blast searches supported at the HumanOlfactory Data Explorer (e.g.,https://genome.weizmann.ac.il/cgi-bin/horde/blastHorde.pl); inparticular for amino acid identity, those using BLASTP 2.2.28+ with thefollowing parameters: Matrix: BLOSUM62; Gap Penalties: Existence: 11,Extension: 1; Neighboring words threshold: 11; Window for multiple hits:40.

In the context of the present invention it shall be understood that anyembodiments referred to as “comprising” certain features of theinvention, shall be understood to include in some more preferredembodiments the more restricted description of “consisting of” or“consisting essentially of” the very same features of the presentinvention.

In another additional or alternative embodiment, the antigen recognizingconstruct may further comprise a CDR1 and/or a CDR2 domain sequence.Within the variable domain, CDR1 and CDR2 are found in the variable (V)region of a polypeptide chain, and CDR3 includes some of V, all ofdiversity (D) and joining (J) regions. CDR3 is the most variable and isthe main CDR responsible for specifically and selectively recognizing anantigen. CDR1 and CDR2 sequences may be selected from a CDR sequence ofa human variable chain allele.

Native alpha-beta heterodimeric TCRs have an alpha chain and a betachain. Each chain comprises variable, joining and constant regions, andthe beta chain also usually contains a short diversity region betweenthe variable and joining regions, but this diversity region is oftenconsidered as part of the joining region. Each variable region comprisesthree CDRs (Complementarity Determining Regions) embedded in a frameworksequence, one being the hypervariable region named CDR3. There areseveral types of alpha chain variable (Va) regions and several types ofbeta chain variable (Vβ) regions distinguished by their framework, CDR1and CDR2 sequences, and by a partly defined CDR3 sequence. The Va typesare referred to in IMGT nomenclature by a unique TRAV number, Vβ typesare referred to by a unique TRBV number. For more information onimmunoglobulin antibody and TCR genes see the internationalImMunoGeneTics information System®, Lefranc M-P et al (Nucleic AcidsRes. 2015 January; 43(Database issue):D413-22; andhttp://www.imgt.org/).

Therefore, in one additional or alternative embodiment the antigenrecognizing construct of the invention comprises CDR1, CDR2 and CDR3sequences in a combination as provided in table 1 herein below, whichdisplay the respective variable chain allele together with the CDR3sequence. Therefore, preferred are antigen recognizing constructs of theinvention which comprise at least one, preferably, all three CDRsequences CDR1, CDR2 and CDR3. Preferably, an antigen recognizingconstruct of the invention comprises the respective CDR1 to CDR3 of oneindividual herein disclosed TCR variable region of the invention (seetable 1 herein below and the example section).

The term “specificity” or “antigen specificity” or “specific for” agiven antigen, as used herein means that the antigen recognizingconstruct can specifically bind to said antigen, preferably a TAAantigen, more preferably with high avidity, when said antigen ispresented by HLA, preferably by HLA A2. For example, a TCR, as antigenrecognizing construct, may be considered to have “antigenic specificity”for the TAA, if T cells expressing the TCR secrete at least 200 pg/ml ormore (e.g., 250 pg/ml or more, 300 pg/ml or more, 400 pg/ml or more, 500pg/ml or more, 600 pg/ml or more, 700 pg/ml or more, 1000 pg ml or more,2,000 pg/ml or more, 2,500 pg/ml or more, 5,000 pg/ml or more) ofinterferon β (IFN-γ) upon co-culture with HLA A2-positive target cellspulsed with a low concentration of a TAA antigen, such as the TAAepitopes and antigens provided herein below (e.g., about 10-11 mol/l,10-10 mol/l, 10-9 mol/l, 10-8 mol/l, 10-7 mol/l, 10-6 mol/l, 10-5mol/l). Alternatively, or additionally, a TCR may be considered to have“antigenic specificity” for the TAA, if T cells expressing the TCRsecrete at least twice as much IFN-γ as the untransduced backgroundlevel of IFN-γ upon co-culture with target cells pulsed with a lowconcentration of the TAA antigens. Such a “specificity” as describedabove can—for example—be analyzed with an ELISA.

In one alternative or additional embodiment of the invention, theantigen recognizing construct selectively binds to a TAA derivedantigenic peptide; preferably wherein the TAA antigenic peptide is aprotein epitope or peptide having an amino acid sequence shown in SEQ IDNO: 133, or a variant thereof, wherein the variant is an amino aciddeletion, addition, insertion or substitution of not more than three,preferably two and most preferably not more than one amino acidposition. In some embodiments, the antigen recognizing construct of theinvention selectively binds any of the modified MAGEA1-003 peptides setforth in SEQ ID NO: 132 to 142 and 154 to 162. In some preferredembodiments, the antigen recognizing construct of the invention does notselectively bind any of the modified MAGEA1-003 peptides set forth inSEQ ID NO: 143 to 152.

The term “selectivity” or “selective recognizing/binding” is understoodto refer to the property of an antigen recognizing construct, such as aTCR or antibody, to selectively recognize or bind to preferably only onespecific epitope and preferably shows no or substantially nocross-reactivity to another epitope. Preferably “selectivity” or“selective recognizing/binding” means that the antigen recognizingconstruct (e.g. a TCR) selectively recognizes or binds to preferablyonly one specific epitope and preferably shows no or substantially nocross-reactivity to another epitope, wherein said epitope is unique forone protein, such that the antigen recognizing construct shows no orsubstantially no cross-reactivity to another epitope and anotherprotein.

The antigen recognizing construct according to the invention ispreferably selected from an antibody, or derivative or fragment thereof,or a T cell receptor (TCR), or derivative or fragment thereof. Aderivative or fragment of an antibody or TCR of the invention shallpreferably retain the antigen binding/recognizing ability of the parentmolecule, in particular its specificity and/or selectivity as explainedabove. Such binding functionality may be retained by the presence of aCDR3 region as defined herein.

In an embodiment of the invention, the inventive TCRs are able torecognize TAA antigens in a major histocompatibility complex (MHC) classI-dependent manner. “MHC class I-dependent manner,” as used herein,means that the TCR elicits an immune response upon binding to TAAantigens within the context of an MHC class I molecule. The MHC class Imolecule can be any MHC class I molecule known in the art, e.g., HLA-Amolecules. In a preferred embodiment of the invention, the MHC class Imolecule is an HLA-A2 molecule.

The invention provides both single chain antigen recognizing constructand double chain recognizing constructs.

In an embodiment, the TCR alpha variable domain has at least onemutation relative to a TCR alpha domain shown in Table 1; and/or the TCRbeta variable domain has at least one mutation relative to a TCR alphadomain shown in Table 1. In an embodiment, a TCR comprising at least onemutation in the TCR alpha variable domain and/or TCR beta variabledomain has a binding affinity for, and/or a binding half-life for, a TAApeptide-HLA molecule complex, which is at least double that of a TCRcomprising the unmutated TCR alpha domain and/or unmutated TCR betavariable domain.

The TCR alpha chains of the present description may further comprise aTCR alpha transmembrane domain and/or a TCR alpha intracellular domain.The TCR beta chains of the present description may further comprise aTCR beta transmembrane domain and/or a TCR beta intracellular domain.

The invention in particular provides a TCR as antigen recognizingconstruct, or fragment or derivative thereof. The TCR preferably is ofhuman, which is understood as being generated from a human TCR locus andtherefore comprising human TCR sequences. Furthermore, the TCR of theinvention may be characterized in that it is of human origin andspecifically recognizes a TAA antigen of the invention.

Another embodiment of the invention additionally or alternativelyprovides the antigen recognizing construct described above, whichinduces an immune response, preferably wherein the immune response ischaracterized by an increase in interferon (IFN) γ levels.

TCRs of the invention may be provided as single chain α or β, or γ andδ, molecules, or alternatively as double chain constructs composed ofboth the α and β chain, or γ and δ chain.

The antigen recognizing construct of the invention may comprise a TCR αor γ chain; and/or a TCR β or δ chain; wherein the TCR α or γ chaincomprises a CDR3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or 100% sequence identity to an amino acid sequence selected from SEQ IDNos. 3, 15, 27, 39, 51, 63, 75, 87, 99 and 111, and/or wherein the TCR βor δ chain comprises a CDR3 having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID Nos. 9, 21, 33, 45, 57, 69, 81, 93, 105 and 117.

Most preferably, in some additional embodiments, wherein the disclosurerefers to antigen recognizing constructs comprising any one, two or allof the CDR1 to CDR3 regions of the herein disclosed TCR chains (seeTable 1), such antigen recognizing constructs may be preferred, whichcomprise the respective CDR sequence of the invention with not more thanthree, two, and preferably only one, modified amino acid residues. Amodified amino acid residue may be selected from an amino acidinsertion, deletion or substitution. Most preferred is that the three,two, preferably only one modified amino acid residue is the first orlast amino acid residue of the respective CDR sequence. If themodification is a substitution then it is preferable in some embodimentsthat the substitution is a conservative amino acid substitution.

If the antigen recognizing construct of the invention is composed of atleast two amino acid chains, such as a double chain TCR, or antigenbinding fragment thereof, the antigen recognizing construct may comprisein a first polypeptide chain the amino acid sequence according to SEQ IDNO: 3, and in a second polypeptide chain the amino acid sequenceaccording to SEQ ID NO: 9; or in a first polypeptide chain the aminoacid sequence according to SEQ ID NO: 15, and in a second polypeptidechain the amino acid sequence according to SEQ ID NO: 21; or in a firstpolypeptide chain the amino acid sequence according to SEQ ID NO: 27,and in a second polypeptide chain the amino acid sequence according toSEQ ID NO: 33; or in a first polypeptide chain the amino acid sequenceaccording to SEQ ID NO: 39, and in a second polypeptide chain the aminoacid sequence according to SEQ ID NO: 45; or in a first polypeptidechain the amino acid sequence according to SEQ ID NO: 51, and in asecond polypeptide chain the amino acid sequence according to SEQ ID NO:57; or in a first polypeptide chain the amino acid sequence according toSEQ ID NO: 63, and in a second polypeptide chain the amino acid sequenceaccording to SEQ ID NO: 69; or in a first polypeptide chain the aminoacid sequence according to SEQ ID NO: 75, and in a second polypeptidechain the amino acid sequence according to SEQ ID NO: 81; or in a firstpolypeptide chain the amino acid sequence according to SEQ ID NO: 87,and in a second polypeptide chain the amino acid sequence according toSEQ ID NO: 93; or in a first polypeptide chain the amino acid sequenceaccording to SEQ ID NO: 99, and in a second polypeptide chain the aminoacid sequence according to SEQ ID NO: 105; or in a first polypeptidechain the amino acid sequence according to SEQ ID NO: 111, and in asecond polypeptide chain the amino acid sequence according to SEQ ID NO:117. Any one of the aforementioned double chain TCR, or antigen bindingfragments thereof, are preferred TCR of the present invention. In someembodiments, the CDR3 of the double chain TCR of the invention may bemutated. Mutations of the CDR3 sequences of SEQ ID NOs. 3, 9, 15, 21,27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111 and 117, asprovided above preferably include a substitution, deletion, addition, orinsertion of not more than three, preferably not more than two, and mostpreferably not more than one amino acid residue. In some embodiments,the first polypeptide chain may be a TCR α or γ chain, and the secondpolypeptide chain may be a TCR β or δ chain. Preferred is thecombination of an αβ or γδ TCR.

The TCR, or the antigen binding fragment thereof, is in some embodimentscomposed of a TCR α and a TCR β chain, or γ and δ chain. Such a doublechain TCR comprises within each chain variable regions, and the variableregions each comprise one CDR1, one CDR2 and one CDR3 sequence. The TCRscomprises the CDR1 to CDR3 sequences as comprised in the variable chainamino acid sequence of SEQ ID NO: 4 and SEQ ID NO: 10 (R26P1A9), or SEQID NO: 16 and SEQ ID NO: 22 (R26P2A6); or SEQ ID NO: 28 and SEQ ID NO:34 (R26P3H1) or SEQ ID NO: 40 and SEQ ID NO: 46 (R35P3A4), or SEQ ID NO:52 and SEQ ID NO: 58 (R37P1C9), or SEQ ID NO: 64 and SEQ ID NO: 70(R37P1H1), or SEQ ID NO: 76 and SEQ ID NO: 82 (R42P3A9), or SEQ ID NO:88 and SEQ ID NO: 94 (R43P3F2), or SEQ ID NO: 100 and SEQ ID NO: 106(R43P3G5), or SEQ ID NO: 112 and SEQ ID NO: 118 (R59P2E7).

Some embodiments of the invention pertain to a TCR, or a fragmentthereof, composed of a TCR α and a TCR β chain, wherein said TCRcomprises the variable region sequences having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to theamino acid sequence selected from the α and β chain according to SEQ IDNO: 4 and 10 respectively, or 16 and 22 respectively; or 28 and 34respectively or 40 and 46 respectively, or 52 and 58 respectively, or 64and 70 respectively, or 76 and 82 respectively, or 88 and 94respectively, or 100 and 106 respectively or 112 and 118 respectively.

The inventive TCRs may further comprise a constant region derived fromany suitable species, such as any mammal, e.g., human, rat, monkey,rabbit, donkey, or mouse. In an embodiment of the invention, theinventive TCRs further comprise a human constant region. In somepreferred embodiments, the constant region of the TCR of the inventionmay be slightly modified, for example, by the introduction ofheterologous sequences, preferably mouse sequences, which may increaseTCR expression and stability.

Some embodiments of the invention pertain to a TCR, or a fragmentthereof, composed of a TCR α and a TCR β chain, wherein said TCRcomprises the constant region having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or preferably 100% sequence identity to an amino acidsequence selected from of the α and β chain according to SEQ ID NO: 5and 11 respectively, or 17 and 23 respectively; or 29 and 35respectively; or 41 and 47 respectively; or 53 and 59 respectively; or65 and 71 respectively; or 77 and 83 respectively; or 89 and 95respectively; or 101 and 107 respectively; or 113 and 119 respectively.

The TCR α or γ chain of the invention may further comprise a CDR1 havingat least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from SEQ ID Nos. 1, 13, 25,37, 49, 61, 73, 85, 97, and 109; and/or a CDR2 having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 2, 14, 26, 38, 50, 62, 74, 86, 98,and 110.

According to the invention the TCR β or δ chain may further comprise aCDR1 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to an amino acid sequence selected from SEQ ID Nos. 7,19, 31, 43, 55, 67, 79, 91, 103 and 115; and/or a CDR2 having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to anamino acid sequence selected from SEQ ID Nos. 8, 20, 32, 44, 56, 68, 80,92, 104 and 116.

The antigen recognizing construct may in a further embodiment comprise abinding fragment of a TCR, and wherein said binding fragment comprisesCDR1 to CDR3, optionally selected from the CDR1 to CDR3 sequences havingthe amino acid sequences of SEQ ID Nos. 1, 2, 3, or 7, 8, 9 or 13, 14,15, or 19, 20, 21, or 25, 26, 27 or 31, 32, 33 or 37, 38, 39 or 43, 44,45 or 49, 50, 51 or 55, 56, 57 or 61, 62, 63 or 67, 68, 69 or 73, 74, 75or 79, 80, 81 or 85, 86, 87 or 91, 92, 93 or 97, 98, 99 or 103, 104, 105or 109, 110, 111 or 115, 116, 117.

In further embodiments of the invention the antigen recognizingconstruct as described herein elsewhere is a TCR, or a fragment thereof,composed of at least one TCR α and one TCR β chain sequence, whereinsaid TCR α chain sequence comprises the CDR1 to CDR3 sequences havingthe amino acid sequences of SEQ ID NO: 1 to 3, and said TCR β chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 7 to 9; or wherein said TCR α chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 13 to 15, and said TCR β chain sequence comprises the CDR1 toCDR3 sequences having the amino acid sequences of SEQ ID NO: 19 to 21;or wherein said TCR α chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NO: 25 to 27, andsaid TCR β chain sequence comprises the CDR1 to CDR3 sequences havingthe amino acid sequences of SEQ ID NO: 31 to 33; or wherein said TCR αchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NO: 37 to 39, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 43 to 45; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 49to 51, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NO: 55 to 57; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 61 to 63, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NO: 67 to 69; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 73 to 75, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 79 to 81; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 85to 87, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NO: 91 to 93; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 97 to 99, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NO: 103 to 105; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 109 to 111, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 115 to 117.

In further embodiments of the invention the antigen recognizingconstruct as described herein before is a TCR, or a fragment thereof,comprising at least one TCR α and one TCR β chain sequence, wherein saidTCR α chain sequence comprises a variable region sequence having theamino acid sequence of SEQ ID No. 4, and wherein said TCR β chainsequence comprises a variable region sequence having the amino acidsequence of SEQ ID No. 10; or wherein said TCR α chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 16, and wherein said TCR β chain sequence comprises avariable region sequence having the amino acid sequence of SEQ ID No.22; or wherein said TCR α chain sequence comprises a variable regionsequence having the amino acid sequence of SEQ ID No. 28, and whereinsaid TCR β chain sequence comprises a variable region sequence havingthe amino acid sequence of SEQ ID No. 34; or wherein said TCR α chainsequence comprises a variable region sequence having the amino acidsequence of SEQ ID No. 40, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 46; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 52, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 58; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 64, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 70; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 76, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 82; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 88, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 94; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 100, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 106; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 112, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 118.

In further embodiments of the invention the antigen recognizingconstruct as described herein before is a TCR, or a fragment thereof,further comprising a TCR constant region having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 5, 11, 17, 23, 29,35, 41, 47, 53, 59,65, 71, 77, 83, 89, 95, 101, 107, 113 and 119, preferably wherein theTCR is composed of at least one TCR α and one TCR β chain sequence,wherein the TCR α chain sequence comprises a constant region having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto an amino acid sequence selected from SEQ ID Nos. 5, 17, 29, 41, 53,65, 77, 89, 101 and 113.

Also disclosed are antigen recognizing constructs as described hereinbefore comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 6, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 12. The invention also provides TCRs comprising afirst TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID No. 18,and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNo. 24. In further embodiments the invention provides antigenrecognizing constructs which are TCR and comprise a first TCR chainhaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID No. 30, and a second TCRchain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID No. 36. Infurther embodiments the invention provides antigen recognizingconstructs which are TCR and comprise a first TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 42, and a second TCR chain having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto the amino acid sequence of SEQ ID No. 48. In further embodiments theinvention provides antigen recognizing constructs which are TCR andcomprise a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNo. 54, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID No. 60. In further embodiments the invention provides antigenrecognizing constructs which are TCR and comprise a first TCR chainhaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID No. 66, and a second TCRchain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID No. 72. Infurther embodiments the invention provides antigen recognizingconstructs which are TCR and comprise a first TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 78, and a second TCR chain having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto the amino acid sequence of SEQ ID No. 84. In further embodiments theinvention provides antigen recognizing constructs which are TCR andcomprise a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNo. 90, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID No. 96. In further embodiments the invention provides antigenrecognizing constructs which are TCR and comprise a first TCR chainhaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID No. 102, and a second TCRchain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID No. 108. Infurther embodiments the invention provides antigen recognizingconstructs which are TCR and comprise a first TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 114, and a second TCR chain having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto the amino acid sequence of SEQ ID No. 120.

As used herein, the term “murine” or “human,” when referring to anantigen recognizing construct, or a TCR, or any component of a TCRdescribed herein (e.g., complementarity determining region (CDR),variable region, constant region, α chain, and/or β chain), means a TCR(or component thereof), which is derived from a mouse or a humanunrearranged TCR locus, respectively.

In an embodiment of the invention, chimeric TCR are provided, whereinthe TCR chains comprise sequences from multiple species. Preferably, aTCR of the invention may comprise an a chain comprising a human variableregion of an α chain and, for example, a murine constant region of amurine TCR α chain.

In one embodiment, the TCR of the invention is a human TCR comprisinghuman variable regions according to the above embodiments and humanconstant regions.

In some embodiments the antigen recognizing construct is murinized orhumanized. These terms are used when amino acid sequences from a foreignspecies are introduced into a construct of the invention.

The TCR of the invention may be provided as a single chain TCR (scTCR).A scTCR can comprise a polypeptide of a variable region of a first TCRchain (e.g., an alpha chain) and a polypeptide of an entire(full-length) second TCR chain (e.g., a beta chain), or vice versa.Furthermore, the scTCR can optionally comprise one or more linkers whichjoin the two or more polypeptides together. The linker can be, forinstance, a peptide, which joins together two single chains, asdescribed herein. Also provided is such a scTCR of the invention, whichis fused to a human cytokine, such as IL-2, IL-7 or IL-15.

The antigen recognizing construct according to the invention can also beprovided in the form of a multimeric complex, comprising at least twoscTCR molecules, wherein said scTCR molecules are each fused to at leastone biotin moiety, or other interconnecting molecule/linker, and whereinsaid scTCRs are interconnected by biotin-streptavidin interaction toallow the formation of said multimeric complex. Similar approaches knownin the art for the generation of multimeric TCR are also possible andincluded in this disclosure. Also provided are multimeric complexes of ahigher order, comprising more than two scTCR of the invention.

For the purposes of the present invention, a TCR is a moiety having atleast one TCR alpha or gamma and/or TCR beta or delta variable domain.Generally, they comprise both a TCR alpha variable domain and a TCR betavariable domain, alternatively both a TCR gamma variable domain and aTCR delta variable domain. They may be αβ/γδ heterodimers or may be insingle chain format. For use in adoptive therapy, an αβ or γδheterodimeric TCR may, for example, be transfected as full length chainshaving both cytoplasmic and transmembrane domains. If desired, anintroduced disulfide bond between residues of the respective constantdomains may be present.

In a preferred embodiment, the antigen recognizing construct is a humanTCR, or fragment or derivative thereof. A human TCR or fragment orderivative thereof is a TCR, which comprises over 50% of thecorresponding human TCR sequence. Preferably, only a small part of theTCR sequence is of artificial origin or derived from other species. Itis known, however, that chimeric TCRs, e.g. derived from human originwith murine sequences in the constant domains, are advantageous.Particularly preferred are, therefore, TCRs in accordance with thepresent invention, which contains murine sequences in the extracellularpart of their constant domains.

Thus, it is also preferred that the inventive antigen recognizingconstruct is able to recognize its antigen in a human leucocyte antigen(HLA) dependent manner, preferably in a HLA-A02 dependent manner. Theterm “HLA dependent manner” in the context of the present inventionmeans that the antigen recognizing construct binds to the antigen onlyin the event that the antigenic peptide is presented by said HLA.

The antigen recognizing construct in accordance with the invention inone embodiment preferably induces an immune response, preferably whereinthe immune response is characterized by the increase in interferon (IFN)γ levels.

Also provided by the invention is a polypeptide comprising a functionalportion of any of the TCRs (or functional variants thereof) describedherein, for examples, of any one of the TCRs selected from R26P1A9,R26P2A6, R26P3H1, R35P3A4, R37P1C9, R37P1H1, R42P3A9, R43P3F2, R43P3G5and R59P2E7, as provided in the example section and table 1. The term“polypeptide” as used herein includes oligopeptides and refers to asingle chain of amino acids connected by one or more peptide bonds. Withrespect to the inventive polypeptides, the functional portion can be anyportion comprising contiguous amino acids of the TCR (or functionalvariant thereof), of which it is a part, provided that the functionalportion specifically binds to the TAA antigen, preferably as disclosedherein in Table 2, and peptides MAGEA1-003_A1 to A9 (SEQ ID NOs:134-142) and MAGEA1-003_T1 to T9 (SEQ ID NOs: 154-162). The term“functional portion” when used in reference to a TCR (or functionalvariant thereof) refers to any part or fragment of the TCR (orfunctional variant thereof) of the invention, which part or fragmentretains the biological activity of the TCR (or functional variantthereof), of which it is a part (the parent TCR or parent functionalvariant thereof). Functional portions encompass, for example, thoseparts of a TCR (or functional variant thereof) that retain the abilityto specifically bind to the TAA antigen (in an HLA dependent manner), ordetect, treat, or prevent cancer, to a similar extent, the same extent,or to a higher extent, as the parent TCR (or functional variantthereof). In reference to the parent TCR (or functional variantthereof), the functional portion can comprise, for instance, about 10%,25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent TCR variablesequences (or functional variant thereof).

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, in whichadditional amino acids are not found in the amino acid sequence of theparent TCR or functional variant thereof. Desirably, the additionalamino acids do not interfere with the biological function of thefunctional portion, e.g., specifically binding to the TAA antigens;and/or having the ability to detect cancer, treat or prevent cancer,etc. More desirably, the additional amino acids enhance the biologicalactivity, as compared to the biological activity of the parent TCR orfunctional variant thereof.

The polypeptide can comprise a functional portion of either or both ofthe α and β chains of the TCRs or functional variant thereof of theinvention, such as a functional portion comprising one of more of CDR1,CDR2, and (preferably) CDR3 of the variable region(s) of the α chainand/or β chain of a TCR or functional variant thereof of the invention.In an embodiment of the invention, the polypeptide can comprise afunctional portion comprising the amino acid sequence of SEQ ID NO: 3,9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111and 117 (CDR3 of the variable regions of the TCR of the invention), or acombination thereof. In an embodiment of the invention, the inventivepolypeptide can comprise, for instance, the variable region of theinventive TCR or functional variant thereof comprising a combination ofthe CDR regions set forth above. In this regard, the polypeptide cancomprise the amino acid sequence of any of SEQ ID NO: 4, 10, 16, 22, 28,34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112 and 118 (thevariable regions of an α or β chain of the TCR of the invention).

In some instances, the construct of the invention may comprise one ortwo polypeptide chains comprising sequences according to any of the SEQID NO: 1 to 120 (CDR sequences, constant and variable regions andfull-length sequences), or functional fragments thereof, and furthercomprise(s) other amino acid sequences, e.g., an amino acid sequenceencoding an immunoglobulin or a portion thereof, then the inventiveprotein can be a fusion protein. In this regard, the invention alsoprovides a fusion protein comprising at least one of the inventivepolypeptides described herein along with at least one other polypeptide.The other polypeptide can exist as a separate polypeptide of the fusionprotein, or can exist as a polypeptide, which is expressed in frame (intandem) with one of the inventive polypeptides described herein. Theother polypeptide may include any peptidic or proteinaceous molecule, ora portion thereof, including, but not limited to an immunoglobulin, CD3,CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d,etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. In some embodiments of the invention, theTCRs (and functional portions and functional variants thereof),polypeptides, and proteins of the invention may be expressed as a singleprotein comprising a linker peptide linking the a chain and the β chain,and linking the γ chain and the δ chain. In this regard, the TCRs (andfunctional variants and functional portions thereof), polypeptides, andproteins of the invention comprising the amino acid sequences of thevariable regions of the TCR of the invention and may further comprise alinker peptide. The linker peptide may advantageously facilitate theexpression of a recombinant TCR (including functional portions andfunctional variants thereof), polypeptide, and/or protein in a hostcell. The linker peptide may comprise any suitable amino acid sequence.Linker sequences for single chain TCR constructs are well known in theart. Such a single chain construct may further comprise one, or two,constant domain sequences. Upon expression of the construct includingthe linker peptide by a host cell, the linker peptide may also becleaved, resulting in separated α and β chains, and separated γ and δchain.

The TCR of the invention may be modified in order to avoid mispairing ofthe TCR chains. The term “mispairing” shall relate to the incorrectpairing between a TCR chain of a TCR α/γ or β/δ transgene of theinvention and an endogenous TCR α/γ or β/δ chain, respectively, andresults in diluted cell surface expression of the transgenic TCRαβ/γδheterodimer, which reduces the functional avidity of the modified Tcells. Preferably, Q at position 44 in the TCR variable domain accordingto the IMGT numbering is substituted by another amino acid in one orboth chains of the TCR of the invention. The substitution is preferablyselected from the group consisting of R, D, E, K, I, W and V.

As already mentioned above, the binding functionality of the TCR of theinvention may be provided in the framework of an antibody. For example,CDR sequences of the TCR of the invention, possibly including additional3, 2 or 1 N and/or C terminal framework residues, may be directlygrafted into an antibody variable heavy/light chain sequence. The term“antibody” in its various grammatical forms is used herein to refer toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain anantigen-binding site or a paratope. Such molecules are also referred toas “antigen binding fragments” of immunoglobulin molecules. Theinvention further provides an antibody, or antigen binding portionthereof, which specifically binds to the antigens described herein. Theantibody can be any type of immunoglobulin that is known in the art. Forinstance, the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG,IgM, etc. The antibody can be monoclonal or polyclonal. The antibody canbe a naturally-occurring antibody, e.g., an antibody isolated and/orpurified from a mammal, e.g., mouse, rabbit, goat, horse, chicken,hamster, human, etc. Alternatively, the antibody can be agenetically-engineered antibody, e.g., a humanized antibody or achimeric antibody. The antibody can be in monomeric or polymeric form.

The term “antibody” includes, but is not limited to, geneticallyengineered or otherwise modified forms of immunoglobulins, such asintrabodies, chimeric antibodies, fully human antibodies, humanizedantibodies (e.g. generated by “CDR-grafting”), antibody fragments, andheteroconjugate antibodies (e.g., bispecific antibodies, diabodies,triabodies, tetra-bodies, etc.). The term “antibody” includescys-diabodies and minibodies. Thus, each and every embodiment providedherein in regard to “antibodies”, or “antibody like constructs” is alsoenvisioned as, bi-specific antibodies, diabodies, scFv fragments,chimeric antibody receptor (CAR) constructs, diabody and/or minibodyembodiments, unless explicitly denoted otherwise. The term “antibody”includes a polypeptide of the immunoglobulin family or a polypeptidecomprising fragments of an immunoglobulin that is capable ofnon-covalently, reversibly, and in a specific manner binding acorresponding antigen, preferably the TAA of the invention, as disclosedherein. An exemplary antibody structural unit comprises a tetramer. Insome embodiments, a full length antibody can be composed of twoidentical pairs of polypeptide chains, each pair having one “light” andone “heavy” chain (connected through a disulfide bond). Antibodystructure and isotypes are well known to the skilled artisan (forexample from Janeway's Immunobiology, 9th edition, 2016).

The recognized immunoglobulin genes of mammals include the kappa,lambda, alpha, gamma, delta, epsilon, and mu constant region genes, aswell as the myriad immunoglobulin variable region genes (for moreinformation on immunoglobulin genes see the internationalIm-MunoGeneTics information System®, Lefranc M-P et al, Nucleic AcidsRes. 2015 January; 43(Database issue):D413-22; andhttp://www.imgt.org/). For full-length chains, the light chains areclassified as either kappa or lambda. For full-length chains, the heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE,respectively. The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. The terms variable light chain (VL) and variable heavychain (VH) refer to these regions of light and heavy chainsrespectively. As used in this invention, an “antibody” encompasses allvariations of antibody and fragments thereof. Thus, within the scope ofthis concept are full length antibodies, chimeric antibodies, humanizedantibodies, single chain antibodies (scFv), Fab, Fab′, and multimericversions of these fragments (e.g., F(ab′)2) with the same, essentiallythe same or similar binding specificity. In some embodiments, theanti-body binds specifically to a peptide TAA of the invention.Preferred antigen recognizing constructs according to the inventioninclude an antibody heavy chain, preferably the variable domain thereof,or an antigen binding fragment thereof, and/or an antibody light chain,preferably the variable domain thereof, or an antigen binding fragmentthereof. Similarly, disulfide-stabilized variable region fragments(dsFv) can be prepared by recombinant DNA technology, antibody fragmentsof the invention, however, are not limited to these exemplary types ofantibody fragments. Also, the antibody, or antigen binding portionthereof, can be modified to comprise a detectable label, such as, forinstance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles). In some instances, the TCR CDR3 sequence may be slightlymodified, but preferably by not more than 3 amino acid residues,preferably only two and most preferably only one amino acid position, ascompared to the CDR3 sequences provided in SEQ ID Nos: 3, 9, 15, 21, 27,33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111 and 117.Preferably, the antibodies comprise the CDR3, preferably all of CDR1 toCDR3 regions in the combination, as indicated for the TCR of theinvention in table 1, in each case independently, optionally with notmore than three or two, preferably one, amino acid substitution(s),insertion(s) and/or deletion(s) compared to these sequences.

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol, 5, 51 1-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 8 Ed., Garland Publishing, New York, N.Y.(201 1)). Alternatively, other methods, such as EBV-hybridoma methods(Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), andRoder et al, Methods Enzymol, 121, 140-67 (1986)), and bacteriophagevector expression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266.

Some embodiments of the invention also pertain to TCRs, or functionalfragments and polypeptides thereof, which are soluble TCRs. As usedherein, the term “soluble T-cell receptor” refers to heterodimerictruncated variants of native TCRs, which comprise extracellular portionsof the TCR α-chain and β-chain, for example linked by a disulfide bond,but which lack the transmembrane and cytosolic domains of the nativeprotein. The terms “soluble T-cell receptor α-chain sequence and solubleT-cell receptor β-chain sequence” refer to TCR α-chain and β-chainsequences that lack the transmembrane and cytosolic domains. Thesequence (amino acid or nucleic acid) of the soluble TCR α-chain andβ-chains may be identical to the corresponding sequences in a native TCRor may comprise variant soluble TCR α-chain and β-chain sequences, ascompared to the corresponding native TCR sequences. The term “solubleT-cell receptor” as used herein encompasses soluble TCRs with variant ornon-variant soluble TCR α-chain and β-chain sequences. The variationsmay be in the variable or constant regions of the soluble TCR α-chainand β-chain sequences and can include, but are not limited to, aminoacid deletion, insertion, substitution mutations as well as changes tothe nucleic acid sequence, which do not alter the amino acid sequence.Soluble TCR of the invention in any case retain the bindingfunctionality of their parent molecules.

The above problem is further solved by a nucleic acid encoding for anantigen recognizing construct of the invention, or any of theaforementioned protein or polypeptide constructs. The nucleic acidpreferably (a) has a strand encoding for an antigen recognizingconstruct according to the invention; (b) has a strand complementary tothe strand in (a); or (c) has a strand that hybridizes under stringentconditions with a molecule as described in (a) or (b). Stringentconditions are known to the person of skill in the art, specificallyfrom Sambrook et al, “Molecular Cloning”. In addition to that, thenucleic acid optionally has further sequences, which are necessary forexpressing the nucleic acid sequence corresponding to the protein,specifically for expression in a mammalian/human cell. The nucleic acidused can be contained in a vector suitable for allowing expression ofthe nucleic acid sequence corresponding to the peptide in a cell.However, the nucleic acids can also be used to transform anantigen-presenting cell, which may not be restricted to classicalantigen-presenting cells, such as dendritic cells, in such a way thatthey themselves produce the corresponding proteins on their cellularsurface.

In some embodiments, the polypeptides of the antigen recognizingconstructs can be encoded by nucleic acids and expressed in vivo or invitro. Thus, in some embodiments, a nucleic acid encoding an antigenrecognizing construct is provided. In some embodiments, the nucleic acidencodes one part or monomer of an antigen recognizing construct of theinvention (for example one of two chains of a TCR of the invention),and/or another nucleic acid encodes another part or monomer of anantigen recognizing construct of the invention (for example the other oftwo chains of the TCR). In some embodiments, the nucleic acid encodestwo or more antigen recognizing construct polypeptide chains, forexample, at least 2 TCR chains. Nucleic acids encoding multiple antigenrecognizing construct chains can include nucleic acid cleavage sitesbetween at least two chain sequences, can encode transcription ortranslation start site between two or more chains sequences, and/or canencode proteolytic target sites between two or more antigen recognizingconstruct chains.

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication. The nucleic acid can compriseany nucleotide sequence, which encodes any of the TCRs, polypeptides, orproteins, or functional portions or functional variants thereofdescribed herein.

Furthermore, the invention provides a vector comprising a nucleic acidin accordance to the invention as described above. Desirably, the vectoris an expression vector or a recombinant expression vector. The term“recombinant expression vector” refers in context of the presentinvention to a nucleic acid construct that allows for the expression ofan mRNA, protein or polypeptide in a suitable host cell. The recombinantexpression vector of the invention can be any suitable recombinantexpression vector, and can be used to transform or transfect anysuitable host. Suitable vectors include those designed for propagationand expansion or for expression or both, such as plasmids and viruses.Examples of animal expression vectors include pEUK-CI, pMAM, andpMAMneo. Preferably, the recombinant expression vector is a viralvector, e.g., a retroviral vector. The recombinant expression vectorcomprises regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost cell (e.g., bacterium, fungus, plant, or animal), into which thevector is to be introduced and in which the expression of the nucleicacid of the invention may be performed. Furthermore, the vector of theinvention may include one or more marker genes, which allow forselection of transformed or transfected hosts. The recombinantexpression vector can comprise a native or normative promoter operablylinked to the nucleotide sequence encoding the constructs of theinvention, or to the nucleotide sequence, which is complementary to orwhich hybridizes to the nucleotide sequence encoding the constructs ofthe invention. The selections of promoters include, e.g., strong, weak,inducible, tissue-specific and developmental-specific promoters. Thepromoter can be a non-viral promoter or a viral promoter. The inventiverecombinant expression vectors can be designed for either transientexpression, for stable expression, or for both. Also, the recombinantexpression vectors can be made for constitutive expression or forinducible expression.

The invention also pertains to a host cell comprising an antigenrecognizing construct in accordance with the invention. Specifically,the host cell of the invention comprises a nucleic acid, or a vector asdescribed herein above. The host cell can be a eukaryotic cell, e.g.,plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g.,bacteria or protozoa. The host cell can be a cultured cell or a primarycell, i.e., isolated directly from an organism, e.g., a human. The hostcell can be an adherent cell or a suspended cell, i.e., a cell thatgrows in suspension. For purposes of producing a recombinant TCR,polypeptide, or protein, the host cell is preferably a mammalian cell.Most preferably, the host cell is a human cell. While the host cell canbe of any cell type, can originate from any type of tissue, and can beof any developmental stage, the host cell preferably is a peripheralblood leukocyte (PBL) or a peripheral blood mononuclear cell (PBMC).More preferably, the host cell is a T cell. The T cell can be any Tcell, such as a cultured T cell, e.g., a primary T cell, or a T cellfrom a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cellobtained from a mammal, preferably a T cell or T cell precursor from ahuman patient. If obtained from a mammal, the T cell can be obtainedfrom numerous sources, including but not limited to blood, bone marrow,lymph node, the thymus, or other tissues or fluids. T cells can also beenriched for or purified. Preferably, the T cell is a human T cell. Morepreferably, the T cell is a T cell isolated from a human. The T cell canbe any type of T cell and can be of any developmental stage, includingbut not limited to, CD4-positive and/or CD8-positive, CD4-positivehelper T cells, e.g., Th1 and Th2 cells, CD8-positive T cells (e.g.,cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells,naive T cells, and the like. Preferably, the T cell is a CD8-positive Tcell or a CD4-positive T cell.

Preferably, the host cell of the invention is a lymphocyte, preferably,a T lymphocyte, such as a CD4-positive or CD8-positive T-cell. The hostcell furthermore preferably is a tumor reactive T cell specific for TAAexpressing tumor cells.

The objective of the invention is also solved by a method ofmanufacturing a TAA specific antigen recognizing construct, or of a TAAspecific antigen recognizing construct expressing cell line, comprising

-   a. Providing a suitable host cell,-   b. Providing a genetic construct comprising a coding sequence    encoding for an antigen recognizing construct according to the    herein disclosed invention,-   c. Introducing into said suitable host cell said genetic construct,    and-   d. Expressing said genetic construct by said suitable host cell.

The method may further comprise a step of cell surface presentation ofsaid antigen recognizing construct on said suitable host cell.

In other preferred embodiments, the genetic construct is an expressionconstruct comprising a promoter sequence operably linked to said codingsequence.

Preferably, said antigen recognizing construct is of mammalian origin,preferably of human origin. The preferred suitable host cell for use inthe method of the invention is a mammalian cell, such as a human cell,in particular a human T lymphocyte. T cells for use in the invention aredescribed in detail herein above.

Also encompassed by the invention are embodiments, wherein said antigenrecognizing construct is a modified TCR, wherein said modification isthe addition of functional domains, such as a label or a therapeuticallyactive substance. Furthermore, encompassed are TCR having alternativedomains, such as an alternative membrane anchor domain instead of theendogenous transmembrane region.

Desirably, the transfection system for introducing the genetic constructinto said suitable host cell is a retroviral vector system. Such systemsare well known to the skilled artisan.

Also comprised by the present invention is in one embodiment theadditional method step of isolation and purification of the antigenrecognizing construct from the cell and, optionally, the reconstitutionof the translated antigen recognizing construct-fragments in a T-cell.

In an alternative aspect of the invention a T-cell is provided obtainedor obtainable by a method for the production of a T cell receptor (TCR),which is specific for tumorous cells and has high avidity as describedherein above. Such a T cell is depending on the host cell used in themethod of the invention, for example, a human or non-human T-cell,preferably a human TCR.

The term “isolated” as used herein in the context of a polypeptide, suchas an antigen recognizing construct (an example of which could be anantibody), refers to a polypeptide that is purified from proteins orpolypeptides or other contaminants that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use. An antigenrecognizing construct according to the invention may be a recombinant,synthetic or modified (non-natural) antigen binding construct. The term“isolated” as used herein in the context of a nucleic acid or cellsrefers to a nucleic acid or cells that is/are purified from DNA, RNA,proteins or polypeptides or other contaminants (such as other cells)that would interfere with its therapeutic, diagnostic, prophylactic,research or other use, or it refers to a recombinant, synthetic ormodified (non-natural) nucleic acid. In this context, a “recombinant”protein/polypeptide or nucleic acid is one made using recombinanttechniques. Methods and techniques for the production of recombinantnucleic acids and proteins are well known in the art.

Treatment Methods and Diseases

One further aspect of the present invention relates to the hereindisclosed antigen recognizing constructs, nucleic acids, vectors,pharmaceutical compositions and/or host cell for use in medicine. Theuse in medicine in one preferred embodiment includes the use in thediagnosis, prevention and/or treatment of a tumor disease, such as amalignant or benign tumor disease. The tumor disease is, for example, atumor disease characterized by the expression of the TAA, in a cancer ortumor cell of said tumor disease.

With respect to the above mentioned medical applications of the antigenrecognizing constructs and other materials derived therefrom, pertainingthereto or encoding the same, in accordance of the present disclosure,the to be treated and/or to be diagnosed diseases can be anyproliferative disorder, preferably characterized by the expression ofthe TAA or TAA epitope sequence of the invention, for example anycancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vagina, cancer ofthe vulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer. A preferred cancer is cancer is cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. Aparticularly preferred cancer is a TAA positive cancer, includingpreferably, melanoma, gastrointestinal and gastric cancer.

The constructs, proteins, TCRs antibodies, polypeptides and nucleicacids of the invention are in particular for use in immune therapy,preferably, in adoptive T cell therapy. The administration of thecompounds of the invention can, for example, involve the infusion of Tcells of the invention into said patient. Preferably, such T cells areautologous T cells of the patient and in vitro transduced with a nucleicacid or antigen recognizing construct of the present invention.

The inventive antigen recognizing constructs, TCRs, polypeptides,proteins (including functional variants thereof), nucleic acids,recombinant expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof),all of which are collectively referred to as “inventive TCR materials”hereinafter, can be formulated into a composition, such as apharmaceutical composition. In this regard, the invention provides apharmaceutical composition comprising any of the antigen recognizingconstructs, TCRs, polypeptides, proteins, functional portions,functional variants, nucleic acids, expression vectors, host cells(including populations thereof), and antibodies (including antigenbinding portions thereof) described herein, and a pharmaceuticallyacceptable carrier, excipient and/or stabilizer. The inventivepharmaceutical compositions containing any of the inventive TCRmaterials can comprise more than one inventive TCR material, e.g., apolypeptide and a nucleic acid, or two or more different TCRs (includingfunctional portions and functional variants thereof). Alternatively, thepharmaceutical composition can comprise an inventive TCR material incombination with another pharmaceutically active agent(s) or drug(s),such as chemotherapeutic agents, e.g., asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, vincristine, etc. Preferably, the carrier is apharmaceutically acceptable carrier. With respect to pharmaceuticalcompositions, the carrier can be any of those conventionally used forthe particular inventive TCR material under consideration. Suchpharmaceutically acceptable carriers are well-known to those skilled inthe art and are readily available to the public. It is preferred thatthe pharmaceutically acceptable carrier be one, which has no detrimentalside effects or toxicity under the conditions of use.

Thus also provided is a pharmaceutical composition, comprising any ofthe herein described products of the invention and TCR materials of theinvention, specifically any proteins, nucleic acids or host cells. In apreferred embodiment the pharmaceutical composition is for immunetherapy, preferably adoptive cell therapy.

Preferably, the inventive TCR material is administered by injection,e.g., intravenously. When the inventive TCR material is a host cellexpressing the inventive TCR (or functional variant thereof), thepharmaceutically acceptable carrier for the cells for injection mayinclude any isotonic carrier such as, for example, normal saline (about0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott,Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5%dextrose in water, or Ringer's lactate. In an embodiment, thepharmaceutically acceptable carrier is supplemented with human serumalbumen.

For purposes of the invention, the amount or dose (e.g., numbers ofcells when the inventive TCR material is one or more cells) of theinventive TCR material administered may be sufficient to affect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the inventive TCRmaterial should be sufficient to bind to a cancer antigen, or detect,treat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive TCR materialand the condition of the animal (e.g., human), as well as the bodyweight of the animal (e.g., human) to be treated.

It is contemplated that the inventive pharmaceutical compositions,antigen recognizing constructs, TCRs (including functional variantsthereof), polypeptides, proteins, nucleic acids, recombinant expressionvectors, host cells, or populations of cells can be used in methods oftreating or preventing cancer, or TAA-positive premalignancy. Theinventive TCRs (and functional variants thereof) are believed to bindspecifically to the TAA of the invention, such that the TCR (or relatedinventive polypeptide or protein and functional variants thereof), whenexpressed by or on a cell, such as a T cell, is able to mediate animmune response against a target cell expressing the TAA of theinvention, preferably presenting TAA peptides via MHC I or II on thesurface of said target cell. In this regard, the invention provides amethod of treating or preventing a condition, in particular cancer, in amammal, comprising administering to the mammal any of the pharmaceuticalcompositions, antigen recognizing constructs, in particular TCRs (andfunctional variants thereof), polypeptides, or proteins describedherein, any nucleic acid or recombinant expression vector comprising anucleotide sequence encoding any of the TCRs (and functional variantsthereof), polypeptides, proteins described herein, or any host cell orpopulation of cells comprising a nucleic acid or recombinant vector,which encodes any of the constructs of the invention (and functionalvariants thereof), polypeptides, or proteins described herein, in anamount effective to treat or prevent the condition in the mammal,wherein the condition is preferably cancer, such as a cancer expressingthe TAA of the invention.

Examples of pharmaceutically acceptable carriers or diluents useful inthe present invention include stabilizers such as SPGA, carbohydrates(e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteinssuch as albumin or casein, protein containing agents such as bovineserum or skimmed milk and buffers (e.g. phosphate buffer).

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof a condition in a mammal. Furthermore, the treatment or preventionprovided by the inventive method can include treatment or prevention ofone or more conditions or symptoms of the condition, e.g., cancer, beingtreated or prevented. For example, treatment or prevention can includepromoting the regression of a tumor. Also, for purposes herein,“prevention” can encompass delaying the onset of the condition, or asymptom or condition thereof.

The present invention also relates to a method of treating cancercomprising administering a TCR, a nucleic acid, or a host cell of thepresent description in combination with at least one chemotherapeuticagent and/or radiation therapy.

Another aspect of the invention further pertains to a method fordetecting a TAA protein, or a complex of MHC and the TAA protein(protein epitope of the TAA), in a (biological) sample—such as oneobtained from a subject or patient—comprising contacting the sample withan antigen recognizing construct specifically binding to said TAApeptide, or to the TAA peptide/MHC complex, and detecting the bindingbetween said antigen recognizing construct and said TAA peptide, or tothe TAA peptide/MHC complex. In some embodiments, the antigenrecognizing construct is a TCR or antibody, or similar constructs, orpreferably the antigen recognizing construct according to the hereindescribed invention. In some embodiments, the (biological) sample is asample of a tumour or a cancer (such as one of those described elsewhereherein) for example a sample comprising tumour or cancer cells.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

-   a) isolating a cell from said subject;-   b) transforming the cell with at least one vector encoding an    antigen recognizing construct of the present invention to produce a    transformed cell;-   c) expanding the transformed cell to produce a plurality of    transformed cells; and-   d) administering the plurality of transformed cells to said subject.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

-   a) isolating a cell from a healthy donor;-   b) transforming the cell with a vector encoding an antigen    recognizing construct of the present invention to produce a    transformed cell;-   c) expanding the transformed cell to produce a plurality of    transformed cells; and-   d) administering the plurality of transformed cells to said subject.

Also provided is a method of detecting cancer in a biological samplecomprising:

-   a) contacting the biological sample with an antigen recognizing    construct of the present description;-   b) detecting binding of the antigen recognizing construct to the    biological sample.

In some embodiments, the method of detecting cancer is carried out invitro, in vivo or in situ.

Also provided is a method of detecting the presence of a condition in amammal. The method comprises (i) contacting a sample comprising one ormore cells from the mammal with any of the inventive TCRs (andfunctional variants thereof), polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of cells,antibodies, or antigen binding portions thereof, or pharmaceuticalcompositions described herein, thereby forming a complex, and detectingthe complex, wherein detection of the complex is indicative of thepresence of the condition in the mammal, wherein the condition iscancer, such as a TAA expressing malignancy.

With respect to the inventive method of detecting a condition in amammal, the sample of cells can be a sample comprising whole cells,lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive antigen recognizingconstructs (and functional variants thereof), polypeptides, proteins,nucleic acids, recombinant expression vectors, host cells, populationsof cells, or antibodies or TCRs, or antigen binding portions thereof,described herein, can be labeled with a detectable label such as, forinstance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

With respect to the above mentioned medical applications of the TCRmaterial of the invention, the to be treated and/or diagnosed cancer canbe any cancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vagina, cancer ofthe vulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer. A preferred cancer is cancer is cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. Aparticularly preferred cancer is a TAA positive cancer, such as skincancer, such as preferably melanoma, gastrointestinal or gastric cancer.

In general, the invention provides a method for treating a subjectsuffering from a tumor or tumor disease comprising the administration ofthe antigen recognizing constructs, nucleic acids, vectors,pharmaceutical compositions and/or host cell as disclosed by the presentinvention. Preferably the subject is a subject in need of such atreatment. The subject in preferred embodiments is a mammalian subject,preferably a human patient, suffering from a tumor or tumor disease,which is TAA-positive.

In view of the disclosure herein it will be appreciated that theinvention furthermore pertains to the following items:

Item 1: An antigen recognizing construct comprising at least onecomplementary determining region (CDR) 3 having at least 50% sequenceidentity to an amino acid sequence selected from SEQ ID NOs. 3, 9, 15,21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111 and117.

Item 2: The antigen recognizing construct according to item 1, whereinsaid antigen recognizing construct is capable of specifically and/orselectively binding to a TAA of the invention antigenic peptide.

Item 3: The antigen recognizing construct according to item 1 or 2,wherein the antigen recognizing construct is an antibody, or derivativeor fragment thereof, or a T cell receptor (TCR), or a derivative orfragment thereof.

Item 4: The antigen recognizing construct according to any one of items1 to 3, wherein said antigen recognizing construct binds to a humanleucocyte antigen (HLA) presented TAA antigenic peptide, wherein saidHLA is optionally type A2.

Item 5: The antigen recognizing construct according to any one of items1 to 4, wherein the construct specifically and/or selectively binds toan epitope having the amino acid sequence selected from SEQ ID NO: 133to 142, preferably to SEQ ID NO: 133.

Item 6: The antigen recognizing construct according to any one of items1 to 5, wherein the construct is an α/β-TCR, or fragment or derivativethereof, or the construct is a γ/δ-TCR, or a fragment or derivativethereof.

Item 7: The antigen recognizing construct according to any one of items1 to 6, characterized in that the construct is of human origin andspecifically and/or selectively recognizes a TAA antigenic peptide.

Item 8: The antigen recognizing construct according to any one of items1 to 7, wherein said antigen recognizing construct is capable ofinducing an immune response in a subject, optionally wherein the immuneresponse is characterized by an increase in interferon (IFN) γ levels.

Item 9: The antigen recognizing construct according to any one of items1 to 8, comprising a TCR α or γ chain; and/or a TCR β or δ chain;wherein the TCR α or γ chain comprises a CDR3 having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 3, 15, 27, 39, 51, 63, 75, 87, 99 and111, and/or wherein the TCR β or δ chain comprises a CDR3 having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto an amino acid sequence selected from SEQ ID Nos. 9, 21, 33, 45, 57,69, 81, 93, 105 and 117.

Item 10: The antigen recognizing construct according to item 9, whereinthe TCR α or γ chain further comprises a CDR1 having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 1, 13, 25, 37, 49, 61, 73, 85, 97 and109; and/or a CDR2 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%,99%, or 100% sequence identity to an amino acid sequence selected fromSEQ ID Nos. 2, 14, 26, 38, 50, 62, 74, 86, 98 and 110.

Item 11: The antigen recognizing construct according to item 9 or 10,wherein the TCR β or δ chain further comprises a CDR1 having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to anamino acid sequence selected from SEQ ID Nos. 7, 19, 31, 43, 55, 67, 79,91, 103 and 115; and/or a CDR2 having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID Nos. 8, 20, 32, 44, 56, 68, 80, 92, 104 and 116.

Item 12: The antigen recognizing construct according to any of items 1to 11, comprising a TCR variable chain region having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 4, 10, 16, 22, 28, 34, 40, 46, 52,58, 64, 70, 76, 82, 88, 94, 100, 106, 112 and 118.

Item 13: The antigen recognizing construct according to any of items 1to 12, wherein the construct is humanized, chimerized and/or murinized.

Item 14: The antigen recognizing construct according to any of items 1to 13, comprising a binding fragment of a TCR, and wherein said bindingfragment comprises CDR1 to CDR3 optionally selected from the CDR1 toCDR3 sequences having the amino acid sequences of SEQ ID Nos. 1, 2, 3,or 7, 8, 9 or 13, 14, 15, or 19, 20, 21, or 25, 26, 27 or 31, 32, 33 or37, 38, 39 or 43, 44, 45 or 49, 50, 51 or 55, 56, 57 or 61, 62, 63 or67, 68, 69 or 73, 74, 75 or 79, 80, 81 or 85, 86, 87 or 91, 92, 93 or97, 98, 99 or 103, 104, 105 or 109, 110, 111 or 115, 116, 117.

Item 15: The antigen recognizing construct according to any of items 1to 14, wherein the construct is a TCR, or a fragment thereof, composedof at least one TCR α and one TCR β chain sequence, wherein said TCR αchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NO: 1 to 3, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 7 to 9; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 13to 15, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NO: 19 to 21; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 25 to 27, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NO: 31 to 33; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 37 to 39, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 43 to 45; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 49to 51, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NO: 55 to 57; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 61 to 63, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NO: 67 to 69; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 73 to 75, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 79 to 81; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 85to 87, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NO: 91 to 93; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 97 to 99, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NO: 103 to 105; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 109 to 111, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 115 to 117.

Item 16: The antigen recognizing construct according to any of items 1to 15, wherein the construct is a TCR, or a fragment thereof, comprisingat least one TCR α and one TCR β chain sequence, wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 4, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 10; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 16, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 22; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 28, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 34; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 40, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 46; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 52, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 58; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 64, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 70; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 76, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 82; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 88, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 94; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 100, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 106; or wherein said TCR α chain sequence comprises avariable region sequence having the amino acid sequence of SEQ ID No.112, and wherein said TCR β chain sequence comprises a variable regionsequence having the amino acid sequence of SEQ ID No. 118.

Item 17: The antigen recognizing construct according to any of items 1to 16, wherein the construct is a TCR, or a fragment thereof, furthercomprising a TCR constant region having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID Nos. 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71,77, 83, 89, 95, 101, 107, 113 and 119, preferably wherein the TCR iscomposed of at least one TCR α and one TCR β chain sequence, wherein theTCR α chain sequence comprises a constant region having at least 50%,60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an aminoacid sequence selected from SEQ ID Nos. 5, 17, 29, 41, 53, 65, 77, 89,101 and 113.

Item 18: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 6, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 12.

Item 19: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 18, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 24.

Item 20: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 30, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 36.

Item 21: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 42, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 48.

Item 22: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 54, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 60.

Item 23: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 66, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 72.

Item 24: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 78, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 84.

Item 25: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 90, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 96.

Item 26: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 102, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 108.

Item 27: The antigen recognizing construct according to any of items 1to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 114, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 120.

Item 28: A nucleic acid encoding for an antigen recognizing constructaccording to any one of items 1 to 27.

Item 29: A vector comprising a nucleic acid according to item 28.

Item 30: A host cell comprising an antigen recognizing constructaccording to any one of items 1 to 27, or a nucleic acid according toitem 28, or a vector according to item 29.

Item 31: The host cell according to item 30, wherein the cell is alymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, morepreferably a CD4 or CD8 positive T-cell.

Item 32: A pharmaceutical composition comprising the antigen recognizingconstruct according to any of items 1 to 27, or the nucleic acidaccording to item 28, or the vector according to item 29, or the hostcell according to item 30 or 31, and a pharmaceutical acceptablecarrier, stabilizer and/or excipient.

Item 33: The antigen recognizing construct according to any one of items1 to 27, or a nucleic acid according to item 28, or a vector accordingto item 29, or a host cell according to item 30 or 31, or thepharmaceutical composition according to item 32, for use in medicine.

Item 34: The antigen recognizing construct, or the nucleic acid, or thevector, or the host cell, or the pharmaceutical composition, for useaccording to item 33, for use in the diagnosis, prevention, and/ortreatment of a proliferative disease, wherein the disease comprises amalignant or benign tumor disease.

Item 35: The antigen recognizing construct, or the nucleic acid, or thevector, or the host cell, or the pharmaceutical composition, for useaccording to item 34, wherein the tumor disease is characterized by theexpression of TAA in a tumor cell of the tumor disease.

Item 36: The antigen recognizing construct, or the nucleic acid, or thevector, or the host cell, or the pharmaceutical composition, for useaccording to any one of items 33 to 35, wherein the use in medicine is ause in immune therapy optionally comprising an adoptive cell transfer,wherein the immune therapy comprises adoptive autologous or heterologousT-cell therapy.

Item 37: A method of manufacturing a TAA specific antigen recognizingconstruct expressing cell line, comprising

-   a. providing a suitable host cell,-   b. providing a genetic construct comprising a coding sequence    encoding the antigen recognizing construct according to any of items    1 to 27,-   c. introducing into said suitable host cell said genetic construct,-   d. expressing said genetic construct by said suitable host cell.

Item 38: The method according to item 37, further comprising cellsurface presentation of said antigen recognizing construct.

Item 39: The method according to item 37 or 38, wherein the geneticconstruct is an expression construct comprising a promoter sequenceoperably linked to said coding sequence.

Item 40: The method according to any one of items 37 to 39, wherein saidantigen recognizing construct is of mammalian origin, preferably ofhuman origin.

Item 41: The method according to any one of items 37 to 40, wherein saidsuitable host cell is a mammalian cell, optionally selected from a humancell or a human T lymphocyte.

Item 42: The method according to any of items 37 to 41, wherein saidantigen recognizing construct is a modified TCR, wherein saidmodification comprises addition of a functional domain comprising alabel, or an alternative domain comprising a membrane anchor domain.

Item 43: The method according to item 42, wherein said antigenrecognizing construct is an alpha/beta TCR, gamma/delta TCR, or a singlechain TCR (scTCR).

Item 44: The method according to any of items 37 to 43, wherein saidgenetic construct is introduced into said suitable host cell byretroviral transfection.

Item 45: The method according to any of items 37 to 44, furthercomprising the isolation and purification of the antigen recognizingconstruct from the suitable host cell and, optionally, reconstitution ofthe antigen recognizing construct in a T-cell.

Item 46: A method of killing target-cells in a patient, in which targetcells aberrantly express TAA, the method comprising administering to thepatient an effective number of T cells expressing a TCR of any of theaforementioned items, the nucleic acid, the expression vector, the hostcell, and/or the pharmaceutical composition of the aforementioned items.

Item 47: The TCR of any of the aforementioned items, wherein the alphachain comprises a TCR alpha variable domain at least 95% identical tothe amino acid sequence of SEQ ID NO: 4; and the beta chain comprises aTCR beta variable domain at least 95% identical to SEQ ID NO: 10, andwherein the TCR specifically binds to a TAA-peptide MHC moleculecomplex.

Item 48: The TCR of any of the aforementioned items having at least onemutation in the alpha chain relative to SEQ ID NO: 4 and/or having atleast one mutation in the beta chain relative to SEQ ID NO: 10, andwherein the TCR has a binding affinity for, and/or a binding half-lifefor, a TAA peptide-HLA molecule complex, which is at least double thatof the unmutated TCR for the same peptide.

Item 49: The TCR of any of the aforementioned items having at least onemutation in the alpha chain relative to SEQ ID NO: 4 and/or having atleast one mutation in the beta chain relative to SEQ ID NO: 10, andwherein the TCR has modified glycosylation compared to the unmutatedTCR.

Item 50: The method of treating of the aforementioned items, wherein theTCR, the nucleic acid, the expression vector, the host cell or thepharmaceutical composition is administered in at least twoadministrations separated by at least 24 hours.

Item 51: The method of item 50, wherein the TCR, the nucleic acid, theexpression vector, the host cell or the pharmaceutical composition isadministered to the subject over a period of days, weeks or months, forexample by local infusion, such as by an infusion pump and/or a cathetersystem.

Item 52: The method of item 51, wherein said local infusion is into asolid tumor, a blood vessel that feeds a solid tumor, and/or the areasurrounding a solid tumor.

Item 52: The treatment method of any of the aforementioned items,wherein the TCR, the nucleic acid, the expression vector, the host cellor the pharmaceutical composition of is administered in a dose of about104 to about 1010 cells per dose.

Item 53: A TCR comprising at least one alpha chain complementaritydetermining region (CDR) selected from the group consisting of an alphachain CDR1, CDR2 and CDR3 of SEQ ID NO: 4 and/or at least one beta chaincomplementarity determining region (CDR) selected from the groupconsisting of a beta chain CDR1, CDR2 and CDR3 of SEQ ID NO: 10, andwherein the TCR specifically binds to a TAA peptide-MHC moleculecomplex.

The present invention will now be further described in the followingexamples with reference to the accompanying figures and sequences,nevertheless, without being limited thereto. For the purposes of thepresent invention, all references as cited herein are incorporated byreference in their entireties.

TABLE 1 TCR sequences of the invention SEQ ID NO: TCR Chain RegionSequence 1 R26P1A9 alpha CDR1 TSINN 2 R26P1A9 alpha CDR2 IRS 3 R26P1A9alpha CDR3 CLIGASGSRLTF 4 R26P1A9 alpha variableMETLLGVSLVILWLQLARVNSQQGEED domain PQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLR VTLDTSKKSSSLLITASRAADTASYFCLIGASGSRLTFGEGTQLTVNP 5 R26P1A9 alpha constantDIQNPDPAVYQLRDSKSSDKSVCLFTDF domain DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 6 R26P1A9 alpha full-lengthMETLLGVSLVILWLQLARVNSQQGEED PQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLR VTLDTSKKSSSLLITASRAADTASYFCLIGASGSRLTFGEGTQLTVNPDIONPDPA VYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSA VAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVI GFRILLLKVAGFNLLMTLRLWSS 7 R26P1A9 beta CDR1SGHDY 8 R26P1A9 beta CDR2 FNNNVP 9 R26P1A9 beta CDR3 CASSYFGWNEKLFF 10R26P1A9 beta variable MGSWTLCCVSLCILVAKHTDAGVIQSPR domainHEVTEMGQEVTLRCKPISGHDYLFWYR QTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYF CASSYFGWNEKLFFGSGTQLSVL 11 R26P1A9 betaconstant EDLNKVFPPEVAVFEPSEAEISHTQKAT domain LVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSVSYQQGVLSATILYEILLGKATLYAVL VSALVLMAMVKRKDF12 R26P1A9 beta full-length MGSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYR QTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYF CASSYFGWNEKLFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCL ATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSAT FWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSY QQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDF 13R26P2A6 alpha CDR1 NSAFQY 14 R26P2A6 alpha CDR2 TY 15 R26P2A6 alpha CDR3CAMSDVSGGYNKLIF 16 R26P2A6 alpha variable MMKSLRVLLVILWLQLSWVWSQQKEVEdomain QDPGPLSVPEGAIVSLNCTYSNSAFQYF MWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATY LCAMSDVSGGYNKLIFGAGTRLAVHP 17 R26P2A6 alphaconstant YIQNPDPAVYQLRDSKSSDKSVCLFTDF domain DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 18 R26P2A6 alpha full-lengthMMKSLRVLLVILWLQLSWVWSQQKEVE QDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDG RFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSDVSGGYNKLIFGAGTRLAVHPYI QNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMD FKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNF QNLSVIGFRILLLKVAGFNLLMTLRLWSS 19 R26P2A6beta CDR1 MNHEY 20 R26P2A6 beta CDR2 SMNVEV 21 R26P2A6 beta CDR3CASTTPDGTDEQFF 22 R26P2A6 beta variable MGPQLLGYVVLCLLGAGPLEAQVTQNPdomain RYLITVTGKKLTVTCSQNMNHEYMSWY RQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYF CASTTPDGTDEQFFGPGTRLTVL 23 R26P2A6 betaconstant EDLKNVFPPEVAVFEPSEAEISHTQKAT domain LVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 24 R26P2A6 beta full-lengthMGPQLLGYVVLCLLGAGPLEAQVTQNP RYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPE GYKVSRKEKRNFPLILESPSPNQTSLYFCASTTPDGTDEQFFGPGTRLTVLEDLK NVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVST DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQ DRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDSRG 25 R26P3H1 alpha CDR1 VSGNPY26 R26P3H1 alpha CDR2 YITG 27 R26P3H1 alpha CDR3 CAVRDMNRDDKIIF 28R26P3H1 alpha variable MASAPISMLAMLFTLSGLRAQSVAQPE domainDQVNVAEGNPLTVKCTYSVSGNPYLFW YVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFC AVRDMNRDDKIIFGKGTRLHILP 29 R26P3H1 alphaconstant NIQNPDPAVYQLRDSKSSDKSVCLFTDF domain DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 30 R26P3H1 alpha full-lengthMASAPISMLAMLFTLSGLRAQSVAQPE DQVNVAEGNPLTVKCTYSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGF EAEFNKSQTSFHLKKPSALVSDSALYFCAVRDMNRDDKIIFGKGTRLHILPNIQNPD PAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS VIGFRILLLKVAGFNLLMTLRLWSS 31 R26P3H1 betaCDR1 LNHDA 32 R26P3H1 beta CDR2 SQIVND 33 R26P3H1 beta CDR3CASSRAEGGEQYF 34 R26P3H1 beta variable MSNQVLCCVVLCFLGANTVDGGITQSPdomain KYLFRKEGQNVTLSCEQNLNHDAMYW YRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYL CASSRAEGGEQYFGPGTRLTVT 35 R26P3H1 betaconstant EDLKNVFPPEVAVFEPSEAEISHTQKAT domain LVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 36 R26P3H1 beta full-lengthMSNQVLCCVVLCFLGANTVDGGITQSP KYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAE GYSVSREKKESFPLTVTSAQKNPTAFYLCASSRAEGGEQYFGPGTRLTVTEDLKN VFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQ DRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDSRG 37 R35P3A4 alpha CDR1 DSASNY38 R35P3A4 alpha CDR2 IRS 39 R35P3A4 alpha CDR3 CAASPTGGYNKLIF 40R35P3A4 alpha variable MTSIRAVFIFLWLQLDLVNGENVEQHPS domainTLSVQEGDSAVIKCTYSDSASNYFPWY KQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAAS PTGGYNKLIFGAGTRLAVHP 41 R35P3A4 alphaconstant YIQNPDPAVYQLRDSKSSDKSVCLFTDF domain DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 42 R35P3A4 alpha full-lengthMTSIRAVFIFLWLQLDLVNGENVEQHPS TLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVT LNKTAKHFSLHITETQPEDSAVYFCAASPTGGYNKLIFGAGTRLAVHPYIQNPDPA VYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSA VAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVI GFRILLLKVAGFNLLMTLRLWSS 43 R35P3A4 betaCDR1 MNHEY 44 R35P3A4 beta CDR2 SVGAGI 45 R35P3A4 beta CDR3CASSLGGASQEQYF 46 R35P3A4 beta variable MSIGLLCCAALSLLWAGPVNAGVTQTPdomain KFQVLKTGQSMTLQCAQDMNHEYMSW YRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSV YFCASSLGGASQEQYFGPGTRLTVT 47 R35P3A4 betaconstant EDLKNVFPPEVAVFEPSEAEISHTQKAT domain LVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 48 R35P3A4 beta full-lengthMSIGLLCCAALSLLWAGPVNAGVTQTP KFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVP NGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSLGGASQEQYFGPGTRLTVTED LKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV STDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDSRG 49 R37P1C9 alpha CDR1TISGTDY 50 R37P1C9 alpha CDR2 G 51 R37P1C9 alpha CDR3 CILFNFNKFYF 52R37P1C9 alpha variable MKLVTSITVLLSLGIMGDAKTTQPNSME domainSNEEEPVHLPCNHSTISGTDYIHWYRQL PSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILFNFNKF YFGSGTKLNVKP 53 R37P1C9 alpha constantNIQNPDPAVYQLRDSKSSDKSVCLFTDF domain DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 54 R37P1C9 alpha full-lengthMKLVTSITVLLSLGIMGDAKTTQPNSME SNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAED RKSSTLILHRATLRDAAVYYCILFNFNKFYFGSGTKLNVKPNIQNPDPAVYQLRDS KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKS DFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLK VAGFNLLMTLRLWSS 55 R37P1C9 beta CDR1 LNHNV56 R37P1C9 beta CDR2 YYDKDF 57 R37P1C9 beta CDR3 CATSSGETNEKLFF 58R37P1C9 beta variable MGPGLLHWMALCLLGTGHGDAMVIQN domainPRYQVTQFGKPVTLSCSQTLNHNVMY WYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAA MYLCATSSGETNEKLFFGSGTQLSVL 59 R37P1C9 betaconstant EDLNKVFPPEVAVFEPSEAEISHTQKAT domain LVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSVSYQQGVLSATILYEILLGKATLYAVL VSALVLMAMVKRKDF60 R37P1C9 beta full-length MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMY WYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAA MYLCATSSGETNEKLFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATL VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFT SVSYQQGVLSATILYEILLGKATLYAVLV SALVLMAMVKRKDF61 R37P1H1 alpha CDR1 TSESNYY 62 R37P1H1 alpha CDR2 QEAY 63 R37P1H1alpha CDR3 CAFGYSGGGADGLTF 64 R37P1H1 alpha variableMTRVSLLWAVVVSTCLESGMAQTVTQS domain QPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATE NRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFGYSGGGADGLTFGKGTHLIIQ P 65 R37P1H1 alpha constantYIQNPDPAVYQLRDSKSSDKSVCLFTDF domain DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 66 R37P1H1 alpha full-lengthMTRVSLLWAVVVSTCLESGMAQTVTQS QPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATE NRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFGYSGGGADGLTFGKGTHLIIQ PYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRS MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN LNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS 67R37P1H1 beta CDR1 SGHDT 68 R37P1H1 beta CDR2 YYEEEE 69 R37P1H1 beta CDR3CASSNEGQGWEAEAFF 70 R37P1H1 beta variable MGPGLLCWALLCLLGAGLVDAGVTQSPdomain THLIKTRGQQVTLRCSPKSGHDTVSWY QQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYL CASSNEGQGWEAEAFFGQGTRLTVV 71 R37P1H1 betaconstant EDLNKVFPPEVAVFEPSEAEISHTQKAT domain LVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSVSYQQGVLSATILYEILLGKATLYAVL VSALVLMAMVKRKDF72 R37P1H1 beta full-length MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWY QQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYL CASSNEGQGWEAEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFT SVSYQQGVLSATILYEILLGKATLYAVLV SALVLMAMVKRKDF73 R42P3A9 alpha CDR1 DSVNN 74 R42P3A9 alpha CDR2 I 75 R42P3A9 alphaCDR3 CAVHNFNKFYF 76 R42P3A9 alpha variable MKRILGALLGLLSAQVCCVRGIQVEQSPdomain PDLILQEGANSTLRCNFSDSVNNLQWF HQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLYISSSQTTDSGVYFCAVHN FNKFYFGSGTKLNVKP 77 R42P3A9 alpha constantNIQNPDPAVYQLRDSKSSDKSVCLFTDF domain DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 78 R42P3A9 alpha full-lengthMKRILGALLGLLSAQVCCVRGIQVEQSP PDLILQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATT VATERYSLLYISSSQTTDSGVYFCAVHNFNKFYFGSGTKLNVKPNIQNPDPAVYQL RDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWS NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL LLKVAGFNLLMTLRLWSS 79 R42P3A9 beta CDR1PRHDT 80 R42P3A9 beta CDR2 FYEKMQ 81 R42P3A9 beta CDR3 CASSLLGQGYNEQFF82 R42P3A9 beta variable MLSPDLPDSAWNTRLLCHVMLCLLGAV domainSVAAGVIQSPRHLIKEKRETATLKCYPIP RHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSS LELGDSALYFCASSLLGQGYNEQFFGP GTRLTVL 83R42P3A9 beta constant EDLKNVFPPEVAVFEPSEAEISHTQKAT domainLVCLATGFYPDHVELSWWVNGKEVHS GVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEND EWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVL VSALVLMAMVKRKDSRG 84 R42P3A9 betafull-length MLSPDLPDSAWNTRLLCHVMLCLLGAV SVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKM QSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLLGQGYNEQFFGP GTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVN GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGK ATLYAVLVSALVLMAMVKRKDSRG 85 R43P3F2 alphaCDR1 TRDTTYY 86 R43P3F2 alpha CDR2 RNSF 87 R43P3F2 alpha CDR3CALSNNNAGNMLTF 88 R43P3F2 alpha variable MLTASLLRAVIASICVVSSMAQKVTQAQdomain TEISVVEKEDVTLDCVYETRDTTYYLFW YKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYF CALSNNNAGNMLTFGGGTRLMVKP 89 R43P3F2 alpha constant HIQNPDPAVYQLRDSKSSDKSVCLFTDF domainDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLN FQNLSVIGFRILLLKVAGFNLLMTLRLWS S 90 R43P3F2alpha full-length MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFW YKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYF CALSNNNAGNMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDS QTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPED TFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 91 R43P3F2 beta CDR1 PRHDT 92 R43P3F2 betaCDR2 FYEKMQ 93 R43P3F2 beta CDR3 CASSPTGTSGYNEQFF 94 R43P3F2 betavariable MLSPDLPDSAWNTRLLCHVMLCLLGAV domainSVAAGVIQSPRHLIKEKRETATLKCYPIP RHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSS LELGDSALYFCASSPTGTSGYNEQFFG PGTRLTVL 95R43P3F2 beta constant EDLKNVFPPEVAVFEPSEAEISHTQKAT domainLVCLATGFYPDHVELSWWVNGKEVHS GVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEND EWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVL VSALVLMAMVKRKDSRG 96 R43P3F2 betafull-length MLSPDLPDSAWNTRLLCHVMLCLLGAV SVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKM QSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSPTGTSGYNEQFFG PGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQF YGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLG KATLYAVLVSALVLMAMVKRKDSRG 97 R43P3G5 alphaCDR1 SSNFYA 98 R43P3G5 alpha CDR2 MTL 99 R43P3G5 alpha CDR3 CALNRDDKIIF100 R43P3G5 alpha variable MEKNPLAAPLLILWFHLDCVSSILNVEQ domainSPQSLHVQEGDSTNFTCSFPSSNFYAL HWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSAT YLCALNRDDKIIFGKGTRLHILP 101 R43P3G5 alphaconstant NIQNPDPAVYQLRDSKSSDKSVCLFTDF domain DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 102 R43P3G5 alpha full-lengthMEKNPLAAPLLILWFHLDCVSSILNVEQ SPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKK GRISATLNTKEGYSYLYIKGSQPEDSATYLCALNRDDKIIFGKGTRLHILPNIQNPD PAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS VIGFRILLLKVAGFNLLMTLRLWSS 103 R43P3G5 betaCDR1 MDHEN 104 R43P3G5 beta CDR2 SYDVKM 105 R43P3G5 beta CDR3CASRLPSRTYEQYF 106 R43P3G5 beta variable MGIRLLCRVAFCFLAVGLVDVKVTQSSRdomain YLVKRTGEKVFLECVQDMDHENMFWY RQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLC ASRLPSRTYEQYFGPGTRLTVT 107 R43P3G5 betaconstant EDLKNVFPPEVAVFEPSEAEISHTQKAT domain LVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 108 R43P3G5 beta full-lengthMGIRLLCRVAFCFLAVGLVDVKVTQSSR YLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEG YSVSREKKERFSLILESASTNQTSMYLCASRLPSRTYEQYFGPGTRLTVTEDLKN VFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQ DRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDSRG 109 R59P2E7 alpha CDR1 DSAIYN110 R59P2E7 alpha CDR2 IQS 111 R59P2E7 alpha CDR3 CAVNSDYKLSF 112R59P2E7 alpha variable METLLGLLILWLQLQWVSSKQEVTQIPA domainALSVPEGENLVLNCSFTDSAIYNLQWFR QDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAV NSDYKLSFGAGTTVTVRA 113 R59P2E7 alphaconstant NIQNPDPAVYQLRDSKSSDKSVCLFTDF domain DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 114 R59P2E7 alpha full-lengthMETLLGLLILWLQLQWVSSKQEVTQIPA ALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNA SLDKSSGRSTLYIAASQPGDSATYLCAVNSDYKLSFGAGTTVTVRANIQNPDPAV YQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV ESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 115 R59P2E7 beta CDR1 PHRDT 116 R59P2E7 beta CDR2FYEKMQ 117 R59P2E7 beta CDR3 CASSLGLGTGDYGYTF 118 R59P2E7 beta variableMLSPDLPDSAWNTRLLCHVMLCLLGAV domain SVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKM QSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLGLGTGDYGYTFG SGTRLTVV 119 R59P2E7 beta constantEDLNKVFPPEVAVFEPSEAEISHTQKAT domain LVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSVSYQQGVLSATILYEILLGKATLYAVL VSALVLMAMVKRKDF120 R59P2E7 beta full-length MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIP RHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSS LELGDSALYFCASSLGLGTGDYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAE ISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAW GRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 121 1G4 alpha CDR1 DSAIYN 122 1G4 alpha CDR2 IQS123 1G4 alpha CDR3 CAVRPTSGGSYIPTF 124 1G4 alpha variableMETLLGLLILWLQLQWVSSKQEVTQIPA domain ALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNA SLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHP 125 1G4 alpha constantYIQNPDPAVYQLRDSKSSDKSVCLFTDF domain DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS S 126 1G4 alpha full-lengthMETLLGLLILWLQLQWVSSKQEVTQIPA ALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNA SLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPYIQNPD PAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS VIGFRILLLKVAGFNLLMTLRLWSS 127 1G4 beta CDR1MNHEY 128 1G4 beta CDR2 SVGAGI 129 1G4 beta CDR3 CASSYVGNTGELFF 130 1G4beta variable MSIGLLCCAALSLLWAGPVNAGVTQTP domainKFQVLKTGQSMTLQCAQDMNHEYMSW YRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSV YFCASSYVGNTGELFFGEGSRLTVL 131 1G4 betaconstant EDLKNVFPPEVAVFEPSEAEISHTQKAT domain LVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 132 1G4 beta full-length MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSW YRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSV YFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLV CLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVS ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSE SYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDSRG

TABLE 2 Peptide sequences of the invention Peptide Code SequenceSEQ ID NO: MAGEA1-003 KVLEYVIKV 133 MAGEA1-003_A1 AVLEYVIKV 134MAGEA1-003_A2 KALEYVIKV 135 MAGEA1-003_A3 KVAEYVIKV 136 MAGEA1-003_A4KVLAYVIKV 137 MAGEA1-003_A5 KVLEAVIKV 138 MAGEA1-003_A6 KVLEYAIKV 139MAGEA1-003_A7 KVLEYVAKV 140 MAGEA1-003_A8 KVLEYVIAV 141 MAGEA1-003_A9KVLEYVIKA 142 TMEM175-001 ILLPYVSKV 143 EPAS-001 KALEGFIAV 144 PTRF-001KLLEKVRKV 145 GALNTL4-001 KLTEYVDKV 146 PSME2-001 KVLERVNAV 147KIAA103-002 KVLNKVITV 148 NSD1-001 KVQEQVHKV 149 TBC1D9-002 LLLPDVIKV150 TMTC4-001 NVLEIVQKV 151 RASAL2-002 SVLEPVISV 152 NYESO1-001SLLMWITQV 153 MAGEA1-003_T1 TVLEYVIKV 154 MAGEA1-003_T2 KTLEYVIKV 155MAGEA1-003_T3 KVTEYVIKV 156 MAGEA1-003_T4 KVLTYVIKV 157 MAGEA1-003_T5KVLETVIKV 158 MAGEA1-003_T6 KVLEYTIKV 159 MAGEA1-003_T7 KVLEYVTKV 160MAGEA1-003_T8 KVLEYVITV 161 MAGEA1-003_T9 KVLEYVIKT 162

EXAMPLES

Ten MAGEA1-003-specific TCRs (R26P1A9, R26P2A6, R26P3H1, R35P3A4,R37P1C9, R37P1H1, R42P3A9, R43P3F2, R43P3G5 and R59P2E7, see Table 1),each encoding tumor specific TCR-alpha and TCR-beta chains, wereisolated and amplified from T-cells of healthy donors. Cells fromhealthy donors were in vitro stimulated according to a method previouslydescribed (Walter et al., 2003 J Immunol., November 15; 171(10):4974-8)and target-specific cells were single-cell sorted using HLA-A*02multimers and then used for subsequent TCR isolation. TCR sequences wereisolated via 5′ RACE by standard methods as described by e.g. MolecularCloning a laboratory manual fourth edition by Green and Sambrook. Thealpha and beta variable regions of TCRs R26P1A9, R26P2A6,R26P3H1R42P3A9, R43P3F2, R43P3G5, R59P2E7 35P3A4, R37P1C9 and R37P1H1were sequenced and expression constructs were generated by genesynthesis for further functional characterization.

R26P1A9, R26P2A6, R26P3H1, R42P3A9, R43P3F2, R43P3G5 and R59P2E7 arederived from HLA-A*02 negative donor (allo-reactive setting) andR35P3A4, R37P1C9 and R37P1H1 are derived from a HLA-A*02 positive donor.

TCRs of interest were expressed in human T cells by transduction, e.g.through mRNA electroporation or lentiviral transduction. For lentiviraltransduction, PBMC were thawed and rested overnight, and then activatedusing immobilized antibodies. Activated cells were transduced using alentiviral vector encoding the MAGEA1-specific TCR and expanded in thepresence of cytokines. T cells were harvested and concentrated bycentrifugation, then cryopreserved.

The T cells were assessed for IFN-γ release after co-culture withdifferent target cells, such as T2 cells loaded with different peptidesas well as tumor cell lines and primary cells from healthy tissues.T-cell activation data are shown in absolute IFNγ levels or a backgroundsubtracted way as indicated below.

Efficacy of CD8+ T cells expressing TCRs R35P3A4, R37P1C9 and R43P3G5was determined e.g. by T cell activation studies (IFNγ release) orkilling assays using different tumor cell lines as target cells. Thecharacterization of the safety profile of TCRs of interest wasapproached by testing the potential activation of TCR-expressing T cellsupon co-culture with isolated primary cell types from healthy tissuesand induced pluripotent stem cell (iPSC)-derived cell types fromHLA-A*02-positive donors (FIG. 33). Cell types were selected in a mannerto cover critical organs like brain, heart and liver and different celltypes as epithelium, endothelium or smooth muscle. Tumor cell lines wereanalyzed side-by-side as positive and negative controls.

Background subtraction method for IFNγ release:Mean_(bg(TCRoi; co))=[mean_((TCRoi; co))−mean_((TCRoi; effector only))]−[mean_((mock; co))−mean_((mock; effector only))]

The respective SD_(bg) was calculated:SD _(bg (TCRoi; co))=[SD _((TCRoi; co)) ² +SD _((TCRoi; effector only))² +SD _((mock;co)) ² +SD _((mock; effector only)) ²]{circumflex over( )}[½]

-   TCRoi=effector cells expressing TCR of interest-   Mock=effector cells without exogenous TCR expression-   Co=effector cells co-cultured with target cells-   Effector only=effector cells not co-cultured-   Mean_((bg))=mean IFNγ release (background subtracted)-   SD_((bg))=standard deviation (background subtracted)

Example 1: T-Cell Receptor R26P1 A9

TCR R26P1A9 (SEQ ID NO:1-12) is restricted towards HLA-A*02-presentedMAGEA1-003 (SEQ ID NO:133) (see FIG. 11). R26P1A9 specificallyrecognizes MAGEA1-003 as human primary CD8+ T-cells re-expressing thisTCR release IFNγ upon co-incubation with HLA-A*02+ target cells loadedeither with MAGEA1-003 peptide or alanine and threonine substitutionvariants of MAGEA1-003 (FIGS. 1 and 22). TCR R26P1A9 does specificallyrecognize MAGEA1-003, but not different peptides showing high degree ofsequence similarity to MAGEA1-003 (FIG. 11). NYESO1-001 peptide is usedas negative control. TCR R26P1A9 has an EC50 of 6 nM (FIG. 36).

Example 2: T-Cell Receptor R26P2A6

TCR R26P2A6 (SEQ ID NO:13-24) is restricted towards HLA-A*02-presentedMAGEA1-003 (SEQ ID NO:133) (see FIG. 12). R26P2A6 specificallyrecognizes MAGEA1-003 as human primary CD8+ T-cells re-expressing thisTCR release IFNγ upon co-incubation with HLA-A*02+ target cells loadedeither with MAGEA1-003 peptide or alanine and threonine substitutionvariants of MAGEA1-003 (FIGS. 2 and 23). TCR R26P2A6 does specificallyrecognize MAGEA1-003, but not different peptides showing high degree ofsequence similarity to MAGEA1-003 (FIG. 12). NYESO1-001 peptide is usedas negative control. TCR R26P2A9 has an EC50 of 100 nM (FIG. 37).

Example 3: T-Cell Receptor R26P3H1

TCR R26P3H1 (SEQ ID NO:25-36) is restricted towards HLA-A*02-presentedMAGEA1-003 (SEQ ID NO:133) (see FIG. 13). R26P3H1 specificallyrecognizes MAGEA1-003 as human primary CD8+ T-cells re-expressing thisTCR release IFNγ upon co-incubation with HLA-A*02+ target cells loadedeither with MAGEA1-003 peptide or alanine and threonine substitutionvariants of MAGEA1-003 (FIGS. 3 and 24). This TCR does specificallyrecognize MAGEA1-003, but not different peptides showing high degree ofsequence similarity to MAGEA1-003 (FIG. 13). NYESO1-001 peptide is usedas negative control. The TCR has an EC50 of 16 nM.

Example 4: T-Cell Receptor R35P3A4

TCR R35P3A4 (SEQ ID NO:37-48) is restricted towards HLA-A*02-presentedMAGEA1-003 (SEQ ID NO:133) (see FIG. 14 above). R35P3A4 specificallyrecognizes MAGEA1-003 as human primary CD8+ T-cells re-expressing thisTCR release IFNγ upon co-incubation with HLA-A*02+ target cells and bindHLA-A*02 tetramers, respectively, loaded either with MAGEA1-003 peptideor alanine and threonine substitution variants of MAGEA1-003 (FIGS. 4and 25). This TCR does specifically recognize MAGEA1-003, but notdifferent peptides showing high degree of sequence similarity toMAGEA1-003 (FIG. 14). NYESO1-001 peptide is used as negative control.The TCR has an EC50 of 16 nM (FIG. 38) and an affinity of 29 μM.

For CD8+ T cells expressing TCR R35P3A4, no activation was observed uponco-culture with HLA-A*02 positive cell types from healthy tissues (seeFIG. 33), while there was an activity towards the tumor cell linesUACC-257 and U266B1 expressing HLA-A*02 and MAGEA1 as source gene forMAGEA1-003 peptide (FIGS. 32 and 33). A corresponding pattern ofreactivity was observed with CD8+ T cells expressing the NYESO1-specificcontrol TCR 1G4, with reactivity towards NYESO1 expressing HLA-A*02positive tumor cell lines but not towards the indicated panel of healthytissue cells.

T-cell activation upon co-culture with cell lines expressing HLA-A*02and MAGEA1 reflects the recognition of endogenously expressed andpresented target pHLA (peptide presented on human leukocyte antigen) byTCRs R35P3A4.

Example 5: T-Cell Receptor R37P1C9

TCR R37P1C9 (SEQ ID NO:49-60) is restricted towards HLA-A*02-presentedMAGEA1-003 (SEQ ID NO:133) (see FIG. 15). R37P1C9 specificallyrecognizes MAGEA1-003 as human primary CD8+ T-cells re-expressing thisTCR release IFNγ upon co-incubation with HLA-A*02+ target cells loadedeither with MAGEA1-003 peptide or alanine and threonine substitutionvariants of MAGEA1-003 (FIGS. 5 and 26). This TCR does specificallyrecognize MAGEA1-003, but not different peptides showing high degree ofsequence similarity to MAGEA1-003 (FIG. 15). NYESO1-001 peptide is usedas negative control. The TCR has an EC50 of 13 nM (FIGS. 34B and 39) andan affinity of 8.7 μM.

Re-expression of R37P1C9 in human primary CD8+ T-cells leads toselective binding of HLA-A*02/MAGEA1-003 tetramers but notHLA-A*02/NYESO1-001 tetramers (FIG. 21). Re-expression of theNYESO1-001-specific TCR 1G4 and mock expression are used as control.

For CD8+ T cells expressing TCR R37P1C9, no activation was observed uponco-culture with HLA-A*02 positive cell types from healthy tissues (seeFIG. 33), while there was an activity towards the tumor cell linesUACC-257 and U266B1 expressing HLA-A*02 and MAGEA1 as source gene forMAGEA1-003 peptide (FIGS. 32 and 33). A corresponding pattern ofreactivity was observed with CD8+ T cells expressing the NYESO1-specificcontrol TCR 1G4, with reactivity towards NYESO1 expressing HLA-A*02positive tumor cell lines but not towards the indicated panel of healthytissue cells.

T-cell activation upon co-culture with cell lines expressing HLA-A*02and MAGEA1 reflects the recognition of endogenously expressed andpresented target pHLA by TCRs R37P1C9, independently from the genedelivery method e.g. mRNA electroporation, lentiviral transduction, etc.(FIGS. 32 and 34).

Example 6: T-Cell Receptor R37P1H1

TCR R37P1H1 (SEQ ID NO: 61-72) is restricted towards HLA-A*02-presentedMAGEA1-003 (SEQ ID NO: 133) (see FIG. 16). R37P1H1 specificallyrecognizes MAGEA1-003 as human primary CD8+ T-cells re-expressing thisTCR release IFNγ upon co-incubation with HLA-A*02+ target cells loadedeither with MAGEA1-003 peptide or alanine and threonine substitutionvariants of MAGEA1-003 (FIGS. 6 and 27). This TCR does specificallyrecognize MAGEA1-003, but not different peptides showing high degree ofsequence similarity to MAGEA1-003 (FIG. 16). NYESO1-001 peptide is usedas negative control. The TCR has an EC50 of 26 nM (FIG. 40).

Re-expression of R37P1H1 in human primary CD8+ T-cells leads toselective binding of HLA-A*02/MAGEA1-003 tetramers but notHLA-A*02/NYESO1-001 tetramers (FIG. 21). Re-expression of theNYESO1-001-specific TCR 1G4 and mock expression are used as control.

Example 7: T-Cell Receptor R42P3A9

TCR R42P3A9 (SEQ ID NO: 73-84) is restricted towards HLA-A*02-presentedMAGEA1-003 (SEQ ID NO: 133) (see FIG. 17). R42P3A9 specificallyrecognizes MAGEA1-003 as human primary CD8+ T-cells re-expressing thisTCR release IFNγ upon co-incubation with HLA-A*02+ target cells loadedeither with MAGEA1-003 peptide or alanine and threonine substitutionvariants of MAGEA1-003 (FIGS. 7 and 28). This TCR does specificallyrecognize MAGEA1-003, but not different peptides showing high degree ofsequence similarity to MAGEA1-003 (FIG. 17). NYESO1-001 peptide is usedas negative control. The TCR has an EC50 of 823 nM (FIG. 41).

Example 8: T-Cell Receptor R43P3F2

TCR R42P3F2 (SEQ ID NO: 85-96) is restricted towards HLA-A*02-presentedMAGEA1-003 (SEQ ID NO: 133) (see FIG. 18). R42P3F2 specificallyrecognizes MAGEA1-003 as human primary CD8+ T-cells re-expressing thisTCR release IFNγ upon co-incubation with HLA-A*02+ target cells loadedeither with MAGEA1-003 peptide or alanine and threonine substitutionvariants of MAGEA1-003 (FIGS. 8 and 29). This TCR does specificallyrecognize MAGEA1-003, but not different peptides showing high degree ofsequence similarity to MAGEA1-003 (FIG. 18). NYESO1-001 peptide is usedas negative control. The TCR has an EC50 of 1,7 nM (FIG. 42).

Example 9: T-Cell Receptor R43P3G5

TCR R43P3G5 (SEQ ID NO: 97-108) is restricted towards HLA-A*02-presentedMAGEA1-003 (SEQ ID NO: 133) (see FIG. 19). R43P3G5 specificallyrecognizes MAGEA1-003 as human primary CD8+ T-cells re-expressing thisTCR release IFNγ upon co-incubation with HLA-A*02+ target cells loadedeither with MAGEA1-003 peptide or alanine and threonine substitutionvariants of MAGEA1-003 (FIGS. 9 and 30). This TCR does specificallyrecognize MAGEA1-003, but not different peptides showing high degree ofsequence similarity to MAGEA1-003 (FIG. 19). NYESO1-001 peptide is usedas negative control. The TCR has an EC50 of 6,6 nM (FIG. 43) and anaffinity of 38 μM.

Re-expression of R43P3G5 in human primary CD8+ T-cells leads toselective binding of HLA-A*02/MAGEA1-003 tetramers but notHLA-A*02/NYESO1-001 tetramers (FIG. 21). Re-expression of theNYESO1-001-specific TCR 1G4 and mock expression are used as control.

For CD8+ T cells expressing TCR R43P3G5, no activation was observed uponco-culture with HLA-A*02 positive cell types from healthy tissues (seeFIG. 33), while there was an activity towards the tumor cell linesUACC-257 and U266B1 expressing HLA-A*02 and MAGEA1 as source gene forMAGEA1-003 peptide (FIGS. 32 and 33). A corresponding pattern ofreactivity was observed with CD8+ T cells expressing the NYESO1-specificcontrol TCR 1G4, with reactivity towards NYESO1 expressing HLA-A*02positive tumor cell lines but not towards the indicated panel of healthytissue cells. T-cell activation upon co-culture with cell linesexpressing HLA-A*02 and MAGEA1 reflects the recognition of endogenouslyexpressed and presented target pHLA by TCRs R43P3G5.

Example 10: T-Cell Receptor R59P2E7

TCR R59P2E7 (SEQ ID NO: 109-120) is restricted towardsHLA-A*02-presented MAGEA1-003 (SEQ ID NO: 133) (see FIG. 20). R59P2E7specifically recognizes MAGEA1-003 as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells loaded either with MAGEA1-003 peptide or alanine andthreonine substitution variants of MAGEA1-003 (FIGS. 10 and 31). ThisTCR does specifically recognize MAGEA1-003, but not different peptidesshowing high degree of sequence similarity to MAGEA1-003 (FIG. 20).NYESO1-001 peptide is used as negative control. The TCR has an EC50 of386 nM (FIG. 44).

Re-expression of R59P2E7 in human primary CD8+ T-cells leads toselective binding of HLA-A*02/MAGEA1-003 tetramers but notHLA-A*02/NYESO1-001 tetramers (FIG. 21). Re-expression of theNYESO1-001-specific TCR 1G4 and mock expression are used as control.

The invention claimed is:
 1. A single chain T cell receptor (TCR)comprising SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, and SEQ ID NO:57, wherein each of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55, and SEQID NO: 57 comprise at most one conservative amino acid substitution. 2.The single chain TCR according to claim 1, wherein said single chain TCRspecifically and/or selectively binds to SEQ ID NO:
 133. 3. The singlechain TCR according to claim 1, comprising a TCR α variable chaincomprising an amino acid sequence having at least 95% identity to SEQ IDNO: 52 and a TCR β variable chain comprising an amino acid sequence atleast having 95% identity to SEQ ID NO:
 58. 4. A nucleic acid encodingfor the single chain TCR according to claim
 1. 5. A vector comprising anucleic acid according to claim
 4. 6. A host cell comprising the singlechain TCR according to claim
 1. 7. A pharmaceutical compositioncomprising the single chain TCR according to claim
 1. 8. A method ofmanufacturing the single chain TCR, comprising providing a suitable hostcell, providing a genetic construct comprising a coding sequenceencoding the single chain TCR according to claim 1, introducing intosaid suitable host cell said genetic construct, expressing said geneticconstruct by said suitable host cell.
 9. The method according to claim8, further comprising isolation and purification of the single chain TCRfrom the suitable host cell and, optionally, reconstitution of thesingle chain TCR in a T-cell.
 10. The single chain TCR of claim 1,comprising a CDR1α chain comprising the amino acid sequence of SEQ IDNO: 49, a CDR2α chain comprising the amino acid sequence of SEQ ID NO:50, a CDR3α chain comprising the amino acid sequence of SEQ ID NO: 51, aCDR1β chain comprising the amino acid sequences of SEQ ID NO: 55, aCDR2β chain comprising the amino acid sequence of SEQ ID NO: 56, and aCDR3β chain comprising the amino acid sequence of SEQ ID NO: 57, whereineach of SEQ ID NOs: 49 and 55 comprise at most one conservative aminoacid substitution.
 11. The single chain TCR of claim 1, comprising aCDR1α chain comprising the amino acid sequence of SEQ ID NO: 49, a CDR2αchain comprising the amino acid sequence of SEQ ID NO: 50, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 51, a CDR1β chaincomprising the amino acid sequences of SEQ ID NO: 55, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 56, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 57, wherein each of SEQID NOs: 50 and 56 comprise at most one conservative amino acidsubstitution.
 12. The single chain TCR of claim 1, comprising a CDR1αchain comprising the amino acid sequence of SEQ ID NO: 49, a CDR2α chaincomprising the amino acid sequence of SEQ ID NO: 50, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 51, a CDR1β chaincomprising the amino acid sequences of SEQ ID NO: 55, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 56, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 57, wherein each of SEQID NOs: 51 and 57 comprise at most one conservative amino acidsubstitution.
 13. The single chain TCR of claim 1, comprising a CDR1αchain comprising the amino acid sequence of SEQ ID NO: 49, a CDR2α chaincomprising the amino acid sequence of SEQ ID NO: 50, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 51, a CDR1β chaincomprising the amino acid sequences of SEQ ID NO: 55, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 56, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO:
 57. 14. The singlechain TCR according to claim 13, wherein said single chain TCRspecifically binds to SEQ ID NO:
 133. 15. The single chain TCR of claim1, wherein the single chain TCR is a soluble TCR.